Abstract:

Abstract:

Abstract:In recent years, marine ranching has become an essential measure for restoring the marine ecological environment and promoting the development of fisheries. The ecological effects of artificial reefs are complex. In addition to providing shelter from predators and living space, artificial reef modules can also affect nutrient transport for vertical water columns by altering the current flow and entraining nutrients, thereby influencing the surrounding biological production available to organisms and proliferating local fishery resources. During the construction process of marine ranching, nutrients are the essential biogenic elements of marine organisms. Their concentration and distribution significantly affect the distribution of fishery resources and anthropogenic activities. Understanding the spatial distribution characteristics of nutrients can provide a reference for adjusting the construction and layout of different functional areas in marine ranching. At present, marine ranching in China mainly focuses on fishery multiplication and leisure industrialization, and the comprehensive utilization efficiency of marine ranching is not high, therefore, allowing the extensive development of marine ranching. However, as an essential part of marine pasture construction, aquaculture activities often lack scientific planning, and exploring the scientific and reasonable layout of aquaculture activities is still in the preliminary stage. This is reflected in the lack of scientific investigations and demonstrations before aquaculture activities. At the same time, the aquaculture varieties are relatively single, leading to poor aquaculture activities. To explore the distribution of nutrients and provide scientific guidance for the development and management of marine ranching, four quarterly surveys were conducted from December 2017 to September 2018 in the marine ranching adjacent to Xiaozhushan Island. Distribution maps of different water layers and profiles of nutrients were generated, and the distribution characteristics were analyzed and discussed. Then, the suitability evaluation method was adopted to explore the suitability of scallop (Patinopecten yessoensis) culturing and deep-sea cages, which are the two main aquaculture activities in marine ranching. Nutrient level index, Chl a concentration, temperature, salinity, water depth, and sediment type were selected as evaluation indexes. The results showed that the seasonal variation of dissolved inorganic phosphorus (DIP) concentration from high to low was winter, summer, autumn, and spring (0.032, 0.021, 0.017, and 0.015 mg/L, respectively). The dissolved inorganic nitrogen (DIN) concentration was seen to decrease seasonally from autumn through winter, summer, and spring (0.26 mg/L, 0.21 mg/L, 0.20 mg/L, and 0.18 mg/L, respectively). The concentrations of DIP and DIN were both significantly different across the four seasons. Still, the distribution was uniform in different seasons. Due to the influence of terrain slope and placement of artificial reefs, the nutrient concentration was higher in the middle and deeper depths close to the area of artificial reef placement. For different seasons, the suitability evaluation results indicated a spatial-temporal instruction in which the suitability index of P. yessoensis culturing was higher in winter and summer. It was prone to be conducted in the west of certain sea areas. However, the suitability index was low in the autumn. Throughout the year, the suitability index of P. yessoensis was higher in the northwest of marine ranching. It was higher in summer and autumn, and it was also suitable for the northwestern region. The suitability index was low in winter. Throughout the year, the suitability index of deep-sea water was higher in the northwest region of marine ranching. One possible reason is the silt sediment in the northwestern area with deep water. A higher suitability index for aquaculture appeared near the area of artificial reef placement for different seasons. Artificial reef is an essential part of the marine ranching area of Xiaozhushan Island and its adjacent areas. However, the placement area of artificial reefs was small, and additionally, the influence of artificial reefs was not included in the suitability evaluation in the present study. As a result, the suitability index of the artificial reef is not high throughout the year, which can be further discussed in the future. Exploring the distribution pattern of water nutrients and evaluating the suitability of aquaculture activities can provide scientific evidence and reference for improvement of stock enhancement, sustainable development, and exploitation of marine ranching. It can also provide a basis for exploring the distribution of marine ranching ecological environments and utilization management.

Abstract:The fishery resources in the Furong Island, Laizhou Bay artificial reef were investigated in 2019; the energy flows and structure of the ecosystem were modeled, and the ecological capacity of Apostichopus japonicus was estimated using an Ecopath with Ecosim 6.6 (EwE 6.6). The model included 16 functional groups that covered the main processes of energy flows in the Furong Island artificial reef ecosystem. According to the results, the trophic levels of the functional groups varied from 1.000 to 3.978; Lateolabrax maculatus occupied the highest trophic level. The total transfer efficiency was 10.6%, and the proportions of the total flow originating from primary producers and detritus were 10.8% and 10.1%, respectively. The total system throughput was estimated to be 2 596.108 t/(km2·a), with 44% originating from detritus. The ratio between the total primary productivity and the total respiration of the system was 1.454; the connectance index was 0.402; the system omnivory index was 0.211; and the Finn cycling index and Finn mean path length were 8.860% and 2.980, respectively. The results showed that the Furong Island artificial reef ecosystem was at a relatively low maturity and stability level with a relatively simple food web. The ecological capacity of A. japonicus estimated by the model was 131 t/km2, which is 6.55 times the existing stock, indicating its growth potential. According to the actual production situation, the ecological balance of the ecosystem can be maintained with an annual catch of 4.1 t/km2. At present, there are few studies using the Ecopath model to evaluate the ecological consequences or ecological capacities of artificial reefs, in China or abroad, and there is little discussion about fishery management models. More applications and verification on artificial reefs are required. This study is expected to provide a scientific basis for the evaluation of ecological consequences of artificial areas and the sustainable utilization of marine ranches.

Abstract:The marine environment has distinct seasonal characteristics in the northwestern South China Sea, under the influence of monsoons, tides, wind stress, and coastal runoff. Decapterus maruadsi is an economically important pelagic fish along the southeast coast and is highly sensitive to the external environment. It is mainly distributed along the coast of the Beibu Gulf and in the waters of western and eastern Guangdong, with abundant resources. To understand the environmental driving mechanisms of D. maruadsi in the northwestern South China Sea, the in-situ fishery data and marine environmental remote sensing data in the northwestern South China Sea from 2012 to 2018 were used to analyze the spatio-temporal distribution of the seasonal average catch per unit effort (CPUE) of D. maruadsi using a generalized additive model. The results showed that the CPUE of D. maruadsi was related to the longitude, sea surface temperature (SST), chlorophyll a (Chl-a) concentration, depth of water, sea surface salinity (SSS), mean direction of total swell, mean direction of wind waves, and mean period of wind waves. The mean direction of wind waves contributed the most to CPUE, followed by the mean direction of the total swell and SST. D. maruadsi occurred mainly in a small area, from longitude 110.5°~114°E, SST 26~30℃, Chl-a 0.2~1.0 mg/m3, depth of water <120 m, SSS 33.4~33.8, mean direction of total swell 70°~120°, 150°~175°, mean direction of wind waves 50°~75°, 120°~135°, 175°~190° and mean period of wind waves 3.0~4.5 s. Moreover, the SST with high CPUE changed significantly withinseasons, contrary to Chl-a. The CPUE of D. maruadsi had obvious seasonal characteristics, with the highest value of 0.848 kg/(kW·d) in summer and the lowest value of 0.087 kg/(kW·d) in winter. The abnormal increase in CPUE in 2016 may have been due to the impact of a super strong El Niño event in 2015–2016. The months in which the sea surface temperature anomaly (SSTA) exceeding 2.5℃ lasted from October 2015 to January 2016, and the spawning period of D. maruadsi is mainly winter and spring, which explains why the CPUE of D. maruadsi began to increase in the winter of 2015. Thus, the super strong El Niño had a positive impact on the replenishment of D. maruadsi in 2016. In this study, the effects of the wind waves and total swell were included in the model for the first time, and we found that wind waves and swells in a certain direction were conducive to the aggregation of D. maruadsi. This may have been due to the south and southwest winds and swells associated with upwelling activity in the northern South China Sea. The changes in the marine environment of the South China Sea are closely related to the ocean dynamics caused by monsoons. To further understand the relationship between the spatio-temporal distribution of D. maruadsi and the marine environment, and to provide a scientific basis for the conservation and adaptive management of D. maruadsi in the northern South China Sea, we can consider adding more representative ocean dynamic factors (such as sea surface and bottom velocities, mixed layer depths, and vertical velocities) to the model, to characterize different dynamic processes and to study the changes in abundance of this fish.

Abstract:Background and aims Nitrogen (N) and phosphorus (P) are the key factors controlling eutrophication, playing an important role in aquatic ecosystems. Currently, eutrophication is severe in almost all major river basins in China, negatively impacting the structure and function of riverine ecosystems. The Changjiang River is one of the most valuable rivers in China. However, enhanced economic development in the Changjiang River basin has caused many adverse environmental problems. To restore the ecological environment of the Changjiang River, a series of decrees and laws were implemented, such as the “10-year fishing moratorium along the Changjiang River.” This lengthy fishing moratorium will almost certainly promote aquaculture in this river basin, likely increasing N and P loadings in the river system and further adversely affecting the watershed-estuarine environment. Owing to the limitations of observations, methodologies, and data, N and P emissions from freshwater aquaculture and their ecological effects are still ambiguous. For this purpose, the total nitrogen (TN) and total phosphorus (TP) emissions caused by freshwater aquaculture in the major provinces along the Changjiang River were estimated using a nutrient transport flux model from 2003 to 2018. The temporal and spatial variations of TN and TP discharges were calculated to determine the potential ecological effects of freshwater aquaculture on riverine material transport and estuarine environments. This work provides a basis for scientific aquaculture planning and environmental management in the Changjiang River basin. This study employs an integrated model to assess the global environment-global nutrient model (IMAGE-GNM), coupling models with hydrology and nutrient delivery, biogeochemistry, and retention. The model describes N and P in feed inputs, fish production, nutrient conversion, and nutrient output. The IMAGE-GNM has already been well applied in the study of N and P sources, retention, and transport in the Changjiang River, demonstrating the model’s credibility in estimating the N and P loads of aquaculture. We evaluated the contributions of N and P and their export to the Changjiang River from 2003 to 2018. (a) Annual emissions of TN and TP caused by freshwater aquaculture in the major provinces along the Changjiang River from 2003 to 2018 were (468.41±179.22) Gg/yr (1 G =109) and (52.02±24.08) Gg/yr, respectively. The emissions of TN in the upper, middle, and lower reaches accounted for 10.44%, 66.38%, and 23.18% of the annual TN emissions, respectively; TP in the upper, middle, and lower reaches accounted for 8.60%, 88.45%, and 24.80%, respectively. The area with the most significant TN and TP emissions caused by freshwater aquaculture was Hubei Province, accounting for (29.96±1.71)% and (31.27±4.11)%, respectively. More than 95% of N and P emissions from freshwater aquaculture were generated by provinces around the main stem of the Changjiang River, especially in the Hubei, Hunan, and Jiangxi Province. (b) The TN emissions from freshwater aquaculture along the Changjiang River increased by 213.96%, from 289.19 Gg/yr in 2003 to 618.76 Gg/yr in 2018. The most significant growth in N emissions was observed in the midstream of the Changjiang River basin from 185.38 Gg/yr to 418.70 Gg/yr. The TP emissions increased from 27.94 Gg/yr in 2003 to 73.55 Gg/yr in 2018. The most significant growth in P emissions was also seen in the midstream from 17.57 Gg/yr to 65.94 Gg/yr. In addition, the growth rates of TN and TP emissions decreased after 2016. The N/P ratio (mass ratio) of freshwater aquaculture discharge in the basin ranged from 6.35 to 12.53 and showed a slowly decreasing trend from 2003 to 2018. (c) According to the model, about 60.52% of the N input through freshwater aquaculture was released in its dissolved form, 20.91% remained in particulate form, and 18.57% was completely converted to biomass and organisms. Approximately 20.93% of the P input through feed was released in the dissolved form, 46.67% remained in the particulate form, and 32.40% was completely converted to biomass. The annual TN and TP emissions of freshwater aquaculture from 2003 to 2018 accounted for an increase of 7.93% and 13.65%, respectively, of the TN and TP emissions in 2010 in the Changjiang River basin. Their increasing trend cannot be underestimated, because the higher biological activity and turnover rates of aquaculture may enhance ecological risks such as eutrophication. Thus, more attention should be paid to the impact of aquaculture activities on the water environment in large river basins. Again, the lower N/P ratio may have a mitigating effect on the imbalance in nutrient stoichiometry in the Changjiang River and its estuary. The change in nutrient origin and delivery would cause a disproportionate increase in non-diatom biomass, potentially breaking the offshore ecosystem stability. Therefore, more attention should be paid to freshwater aquaculture and its environmental effects, and relevant laws and regulations should also be implemented to optimize freshwater aquaculture.

Abstract:The Beibu Gulf is the fourth largest Chinese fishing ground, with rich fishery resources and a significant role in marine fishery production. The feeding ecology of fish is important to explain the structure and function of marine ecosystems. As one of the most important fish species in the Beibu Gulf, Nemipterus japonicus plays an essential role in energy flow and other aspects of the marine ecosystem. Understanding its feeding ecology could provide a basic theoretical basis for the rational utilization and protection of this resource. A total of 248 N. japonicus specimens were collected from Beibu Gulf from January to April and August to December in 2018. The food content composition, feeding intensity, and feeding niche of N. japonicus were analyzed using stomach contents to reflect feeding habits. Combined with carbon and nitrogen stable isotopes, the food source and trophic structure characteristics were analyzed, the trophic level was determined, and the feeding ecology of N. japonicus in the Beibu Gulf was discussed. The stomach content analysis revealed that N. japonicus is a broad-feeding benthic carnivore, which feeds on a large number of species, including eight prey groups, including fish, shrimp, and crab, among which fish and shrimp are the dominant food groups, with a relative importance index of 92.92%. The proportion of food species varied with the season and growth. Fish were an important prey group across all seasons. Moreover, N. japonicus preyed on shrimp in spring, crab in summer, shrimp and polychaetes in autumn, and shrimp and sipunculoidea in winter. With an increase in body length, the feeding habits of N. japonicus gradually change from primarily feeding on shrimp and fish to mainly feeding on fish, and the body length node of feeding transition was approximately 130 mm. There were clear seasonal variations in feeding intensity, with the highest intensity in autumn, followed by that in spring and summer, and the lowest in winter. N. japonicus had a high prey diversity; the Shannon-Wiener diversity index (H') was 1.77, and the Pielou evenness index (Jʹ) was 0.64. From spring to winter, the H' and Jʹ values tended to increase and then decrease, and the highest value occurred in autumn, which meant that the feeding niche was the highest in autumn. The number of prey species increased with individual growth, and Hʹ and Jʹ showed an increasing trend with body length. The group with a body length ≥ 160 mm had the largest niche width, which suggested that the food diversity increased with individual growth, and the diet changed to broad-feeding. Based on stable isotope analysis, δ13C was positively correlated with the body length of N. japonicus (P<0.05), indicating that the trophic source changed with growth. The span of the δ13C (CR) range was 2.86%, indicating extensive basic food sources. The span of the δ15N (NR) range was 4.23%, and the trophic span was more than one trophic level, indicating considerable differences in feeding among individuals. A significant positive correlation was found between body length and δ15N (P<0.01), which indicated that the trophic level increased with an increasing body length. The trophic level of N. japonicus ranged from 3.21 to 4.46, with an average value of 3.97, indicating that N. japonicus species were middle-high carnivorous animals, and were at a high trophic level in the fish food web of the Beibu Gulf. The trophic level obtained in this study is higher than that obtained in other studies, which may be related to the changes in the composition of the prey, N. japonicus has shifted from feeding mainly on the low trophic level (crustaceans) to the high trophic levels (fish). In terms of trophic structure, the CR value of N. japonicus in the Beibu Gulf in spring and winter was lower than that in summer and autumn, indicating that the basic food source diversity in spring and winter is lower than that in summer and autumn, however, there are fewer prey species in summer, which might be caused by the feeding preference of N. japonicus. Simultaneously, the NR values in spring and summer were greater than those in summer and autumn, indicating that the span of the main trophic sources in the feed was higher than that in summer and autumn, consistent with the seasonal variation of dominant prey. Although the standard ellipse area (SEA) overlaps to different degrees among seasons, the total niche (TA) had clear seasonal differences, signifying that there were certain similarities and specific differences in prey resource utilization, which is beneficial for alleviating the seasonal pressure on the dominant prey. Furthermore, the CR value in different body length groups of N. japonicus showed an overall upward trend with individual growth, indicating that the food source diversity increased with growth. The NR value decreased when body length was greater than 130 mm, which indicates that the average trophic level of the population was concentrated when individuals grew to a certain stage. The SEA overlap rate was higher among length groups, implying that the trophic source components were similar at different growth stages; however, TA tended to increase and then decrease with body length, which may reduce food competition within species by niche differentiation.

Abstract:The study of ontogenetic organisms can help us fully understand the law of morphology change and the correlation between the function and morphology of the structure of organisms. To explore the pattern of growth change in U. edulis beak, this study used geometric morphometric methods to analyze the differences in beak size and morphology in different sexes and ontogenetic stages, revealing its ecological implications. Results show some differences in the size of different sexes´ beaks of U. edulis in the ontogenetic process; the beak of female individuals is larger than that of male individuals, but the differences in the beak morphology of different sexes were not significant (P>0.05). Principal component analysis and a thin-plate spline deformation mesh show that the beak morphology from immature to mature presents different developmental stages; in the ontogenetic process, the hood and wing of the upper and lower beak become larger, the crest protrudes more, the jaw angle gradually becomes smaller, and the rostrum of the upper beak is sharper; however, the rostrum of the lower beak gradually blunted. The multiple regression analysis shows that the morphology change of the upper beak is small in the immature stage, the morphology change is larger in the maturing and mature stages, the pattern of morphology change in the maturing and mature stages is similar, the morphology change of the lower beak is large in the immature and mature stages, the morphology change is small in the mature stage, and the morphology change trend is significantly different in the ontogenetic process. The beak morphology of U. edulis had different growth patterns during different ontogenetic stages. Beaks also showed an allometric growth, possibly related to the feeding and environmental changes during the ontogenetic stage of U. edulis, helping it to grow and develop.

Abstract:Coilia nasus is a precious migratory fish typical to the Yangtze River Basin of China. It is locally referred to as the “Three delicious fish of the Yangtze River,” with its fresh meat being particularly delicious and popular. In recent years, wild C. nasus numbers in the Yangtze River Basin have been decreasing due to environmental changes and illegal overfishing, among other reasons. To attenuate the availability of C. nasus, the Shanghai Fisheries Research Institute has successfully established an indoor artificial breeding and pond culture program in 2011. The research and development of C. nasus artificial breeding technologies have steadily progressed, though the artificial breeding yield of C. nasus remains relatively low. In addition, related research on the artificial cultivation of C. nasus is still in its infancy. Research on C. nasus in China and abroad mainly focuses on gonad development, breeding technology, muscle nutrient composition, and growth performance. No reports exist on the effects of brackish water culture conditions on the physiology of C. nasus. Given its economic importance, it would be of great value to evaluate the changes in the digestive, antioxidant, nonspecific immune capacity, and metabolic rates of C. nasus from brackish water (natural seawater in Hangzhou Bay, salinity 8.7~12.5) to freshwater aquaculture, to determine the breeding conditions of C. nasus, and to improve the artificial breeding yield and related technology. To investigate the differences in digestive capacity, antioxidant capacity, nonspecific immune capacity, and metabolic rates of C. nasus in brackish water and freshwater aquaculture conditions, the experiment adopted the pond interbreeding method. Two groups were established: A brackish aquaculture group and a freshwater aquaculture group (control group). Each group was housed in a single aquaculture pond with an area of 0.17 hm2. 500 C. nasus were stocked in each pond and moved to a Takifugu obscurus pond for a seven-month aquaculture experiment. At the end of the experiment, 12 C. nasus were randomly selected from each group and randomly divided into three replicates. Four C. nasus samples were selected from each replicate. The liver, intestine, stomach, and cecum of C. nasus were dissected on an ice plate and carefully removed to prepare a 10% homogenate. The activities of digestive enzymes, antioxidant enzymes, nonspecific immune enzymes, and metabolic enzymes and the total protein content were measured using a kit produced by the Nanjing Jiancheng Bioengineering Institute. The activities of digestive enzymes, antioxidant enzymes, nonspecific immunity enzymes, and metabolic enzymes in brackish water and freshwater aquaculture environments were compared and analyzed in a seven-month aquaculture experiment using the same batch of artificially bred C. nasus. The growth and survival rates of C. nasus cultured in brackish water (hereafter referred to as brackish water C. nasus) were significantly higher than that in the freshwater (hereafter referred to as freshwater C. nasus) (P < 0.05). The digestive ability of protein and starch in brackish water C. nasus was higher than that of freshwater C. nasus, while the digestive ability was weakened. Among them, the activity of amylase (AMS) in the four organs of brackish water C. nasus was higher than those in freshwater, but not significantly so (P>0.05). The activities of protease in the liver and stomach were significantly higher than those in freshwater C. nasus (P < 0.05), and the activities of lipase (LPS) in the liver and cecum were significantly lower than those in freshwater C. nasus (P<0.05). The order of AMS and protease activity of brackish water C. nasus was: cecum > intestine > stomach > liver, while the order of LPS activity was intestine > cecum > stomach > liver. The order of AMS, protease, and LPS activity of freshwater C. nasus was: cecum > intestine > stomach > liver. Brackish water had significant effects on antioxidant, nonspecific immunity, and metabolic abilities, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) in the liver of C. nasus were significantly increased, while the activity of catalase (CAT) was significantly decreased (P<0.05). The activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the liver of C. nasus in brackish water were significantly lower than those in freshwater (P<0.05). Brackish water had no significant effect on the activity of aspartate aminotransferase (AST), but it significantly reduced the activity of alanine aminotransferase (ALT). In summary, brackish water aquaculture conditions promoted the growth of C. nasus; increased the survival rate; improved the ability of C. nasus to digest protein and starch; improved the immunity of fish; and reduced the influence of external stresses by decreasing the activities of AKP, ACP, and ALT enzymes and increasing the activities of SOD and GSH-PX enzymes. This study also found that C. nasus living in brackish water, as a result of maintaining the internal osmotic balance, significantly reduced ALT enzyme activity and reduced urea production and emission, thereby reducing the pollution of the direct environment. Therefore, brackish water is more suitable for the aquaculture of C. nasus, and it is recommended that brackish water (salinity of 8.7~12.5) be used for aquaculture of 1+ age C. nasus individuals in the future. The results of this study provide a scientific basis for the improvement of artificial breeding technology, improving the yield of C. nasus, and the development of special compound feed for C. nasus.

Abstract:Micropterus salmoides is an economically important cultured carnivorous fish in China. In recent years, owing to the development and wide application of artificial formula feed, M. salmoides production has rapidly increased and reached 470 000 tons in 2019. However, a low survival rate of M. salmoides larvae is observed during the first feeding and artificial formula feed adaptation. In this study, to better understand the artificial formula feed adaptation of M. salmoides larvae, the developmental characteristics of the digestive tract and digestive gland of the fish larvae from 2~30 dph were observed and described using histological sections and scanning electron microscopy (SEM). Moreover, the digestive tract (stomach and intestine) characteristics of certain dead larval fish during the first feeding and the transformation of artificial formula feed were investigated. For histological analysis, the larval fish (including stomach, intestine, pyloric cecum, liver, and pancreas tissues) were dehydrated with an alcohol gradient (70%, 80%, 90%, and 100%), embedded in paraffin, cut into 5 μm sections, and stained with standard hematoxylin and eosin. For SEM analysis, the stomach, pyloric cecum, and intestine of 30 dph larval fish were fixed in 2.5% glutaraldehyde solution for 12 h. Then, the tissues were fixed in 1% osmium solution for 2 h, dehydrated with gradient alcohol (70%~100%), soaked in tert-butyl alcohol for 2 h, dried with a lyophilizer, and plated with gold by ion sputtering. Finally, the images were captured using a HITACHIX-650 scanning electron microscope. At a water temperature of (23±1)°C, the larval yolk sac and oil drop gradually decreased at 0~4 dph, the digestive tract was initially differentiated, and heartbeat, blood circulation, mouth crack, esophagus, and anus were observed at 4 dph. Larval fish were in the endogenous nutrition period at this stage. In 4~ 6 dph larval fish, the esophagus, stomach, intestine, liver, and pancreas regions gradually formed, the digestive tube opened to the outside initially, and yolk sac and oil drop significantly decreased and then completely disappeared, indicating that the larval fish entered the endo-exotrophic period. At this stage, sufficient Artemia salina should be provided to induce the larval fish to open their mouth and perform first feeding. In 10~16 dph larval fish, the stomach, pylorus caecum, and intestine were closely arranged. From the surface of the intestinal cavity, the mucosa, submucosa, muscular, and serosa layers were successively presented. The size of the mucosa columnar epithelial cells and the number of goblet cells clearly increased. The masses of hepatocytes and pancreatic cells significantly increased, and a vacuole structure appeared in the hepatocytes. The pancreatic cells were arranged closely, and the blood cells and pancreatic ducts were distributed. At this stage, larval fish entered the exogenous nutrition period. From this stage onward, larval fish are better able to transform feeding artificial formula feed step by step. In 20~24 dph larval fish, the stomach gland was well developed, and the stomach wall thickness and number of stomach glands further increased. Furthermore, the mucosal folds and connective tissue of the submucosa were further differentiated, and the longitudinal mucosal folds increased and curled. In addition, secondary mucosal folds in the intestine gradually appeared and the number of mucosal folds in the pyloric cecum increased. Fatty accumulation and secretory granules were observed in the liver and pancreas, respectively, indicating that the larval hepatopancreas was similar to that of adult fish. The digestive system of larval fish was completely developed at this stage, and the larval fish had the ability to transform and adapt to artificial formula feed. No significant differences were observed in body length and body weight of M. salmoides larvae (P>0.05) during the adaptation to artificial formula feed at 12, 16, and 20 dph. However, the survival rate of 12 dph larval fish (45.59%) was lower than that of 16 (60.60%) and 20 dph larval fish (69.83%). In addition, the body length, body weight, and survival rate of M. salmoides larvae were positively correlated with the time point of feeding artificial formula feed. SEM results showed abundant polygonal reticular mucosal folds in the gastric epidermis of 30-day-old larval fish, and there were dense secretion pores between the gastric pits. Mucosa folds with fixed shapes were observed on the inner surface of the intestine, and mucous cells, secretory pores, and secretory granules were clearly observed between the mucosal folds. Interestingly, the inner surface structure of the pyloric cecum was similar to that of the intestine. A difference was the mucosa folds without a fixed shape on the inner surface of the pyloric caecum. These mucosal folds and secretory pores are important for food digestion, absorption, and excretion, indicating that the larval fish are old enough to adapt to the artificial formula feed at this point. During the first feeding and artificial formula feed adaptation, the stomach and intestine of the larval fish were incompletely developed and accompanied by tissue damage in the dead individuals. For example, the stomach and intestinal cavity were significantly shriveled, the inner epidermis did not have the molding mucous membrane fold and goblet cells, and the mucous membrane layer was cracked or absent. These results indicate that larval fish overfeed and fail to effectively digest, absorb, and expel nutrients, which ultimately results in death associated with nutritional deficiencies, intestinal blockages, and inflammation. During the process of adaptation to artificial formula feed for M. salmoides, the development of larval digestive system was investigated. The digestive system of larval fish at 4~6 dph gradually differentiated and was still in the mixed nutrition period, and sufficient A. salina should be provided to induce the larval fish to open their mouths and perform the first feeding. At 6~16 dph, the digestive system gradually developed and larval fish entered the exogenous nutrition period, and sufficient food could be provided. At 16~20 dph, the digestive system of the larval fish completely developed, and this stage is the optimal time to switch to artificial formula feed. Our study provides basic data for feeding condition optimization of M. salmoides larvae.

Abstract:With the rapid development of the aquaculture industry and the shortage of global fishery resources, the contradiction between supply and demand of fish meal has become increasingly significant. Therefore, finding new substitutes for fish meals and reasonably reducing the amount of fish meal in formula feed has become an important research topic in aquatic feed. Stickwater is a byproduct of fishmeal processing. It contains many water-soluble molecules, such as small molecular peptides, biogenic amines, taurine, and high unsaturated fatty acids. These components are regarded as particular nutrients or bioactive substances in the fish meal. Studies have shown that stickwater can replace part of fish meal in recent years and achieve good results in fish and other aquatic animals. The fermented feed has excellent advantages for fish meal replacement. Studies have shown that after fermentation, the contents of anti-nutritional factors in plant raw materials are decreased. In contrast, the contents of small peptides and free amino acids are increased, the nutrient composition is changed, and the intermediate metabolites of microorganisms are obtained, which can further improve the replacement level of fish meal. There are few studies on the fermentation of feed materials, such as fish meal and stickwater, which do not contain anti-nutritional factors. Our laboratory´s preliminary study found that stickwater combined with other animal and plant proteins could replace 40% of fish meal in the feed. This experiment fermented stickwater first, then replace fish meal in high plant protein formula feed with fermented stickwater (FSW), aims to further increase the amount of fish meal replacement, reduce the negative effect of the plant protein source on the turbot, for FSW application in feeds for turbot provide a theoretical reference. The stickwater in the experiment was a brown viscous liquid, with a water content of 48.73%, dry matter crude protein content of 64.08%, and crude fat content of 8.91%. The strains used were Bacillus subtilis and Lactobacillus. The fermentation conditions were as follows: Temperature of 37℃, the addition of 1% sugar as auxiliary material, Bacillus subtilis and Lactobacillus (1:1) added to 1% of the total mass of the stickwater, and the fermentation period was five days. After fermentation, the content of acid-soluble protein decreased significantly. Still, the crude protein, crude fat, and free amino acids showed no significant changes. It was then used for turbot culture. Healthy juvenile turbot with an average bodyweight of (30.00±0.03) g were randomly divided into six groups with three replicates and 30 fish per replicate. The trial lasted for eight weeks. Six diets consisted of a positive control diet with 50% fish meal (positive control group), a harmful control diet with 30% fish meal (negative control group), and experimental diets formulated by FSW were used to replace 2%, 4%, 6%, and 8% of the fish meal with the harmful control diet, respectively (FSW2, FSW4, FSW6, and FSW8). The results showed: There were no significant differences in the survival rate of juvenile turbot among all groups (P>0.05). There were no significant differences in juvenile turbot´s weight gain rate and protein efficiency ratio in the FSW2~FSW8 group compared with that of the positive control group. Still, they were all higher than those in the negative control group. The crude protein content of whole fish and dorsal muscle in the FSW2~FSW8 group was not significantly different from that in the positive control group. Still, it was significantly higher than that in the negative control group. The highest crude lipid content of whole fish and dorsal muscle was found in the negative control group (P<0.05). Serum ALT, AST, and TG levels in the negative control group were significantly higher than those in the positive control group; the negative control group and FSW2~FSW8 group showed first decreasing and subsequent increasing serum ALT, AST, and T-CHO levels. At the same time, the ALT and AST levels in the liver showed an opposite trend. The serum HDL-C content in the negative control group was significantly lower than that in the positive control group. The activities of PKA, KPC, and LDH in the liver were significantly lower in the negative control group than in the positive control group (P<0.05). Compared with the positive control group, the expression of b0at1 and pept1 in the intestine was upregulated in the negative control group. In contrast, the expression of cat1 and pat1 was not significantly different. The expression levels of boat1, cat1, pat1, and pept1 in the FSW2~FSW8 group were significantly higher than those in the positive control and negative control groups (P<0.05). The results showed that FSW was an excellent substitute for fish meals, and the added amount could be up to 8% in the feed. In the feed with high plant protein, the fish meal can be replaced by FSW to reduce the amount of fish meal further, reduce the metabolic abnormality caused by plant proteins, and improve juvenile turbot´s antioxidant capacity. Under these experimental conditions, the group supplemented with FSW achieved the same growth effect as the positive control group. In conclusion, the fish meal content of juvenile turbot feed can be reduced to 22% by adding FSW without adverse effects on the growth of juvenile turbot. This provides a theoretical reference for the fermentation process of SW and subsequent application of FSW in seawater fish.

Abstract:Rockfish (Sebastes schlegelii) is a near-shore, carnivorous coldwater fish, primarily distributed in the East China Sea, Korean peninsula, Japan, and the Sea of Okhotsk. Owing to its delicious meat, strong disease resistance, rapid growth, and other characteristics, it has become one of the main cultured fish in deep water cages in northern China. A high-quality and efficient formula diet for rockfish has not been developed because of nutrient requirement deficiencies. As vitamin D3 is the most important form of vitamin D and only exists in animals, cultured fish can only obtain vitamin D3 from their diet. Vitamin D3 can maintain the mineral homeostasis in fish, participate in the endocrine system, and affect the proliferation and apoptosis of immune cells. As such, this experiment was conducted to investigate the effects of dietary vitamin D3 on growth performance, body composition, and immune function of juvenile rockfish and determine the optimum dietary vitamin D3 requirement. Six kinds of isonitrogenous and isolipidemic diets with 707, 1254, 1740, 2513, 4519, and 8671 IU/kg of vitamin D3 were prepared by adding 0, 500, 1000, 2000, 4000, and 8000 IU/kg of vitamin D3 into the basic diet. Each diet was randomly assigned to triplicate groups of juveniles with initial bodyweights of (20.95±0.05) g for eight weeks. The fish were challenged with Vibrio harveyi for 48 hours after feeding. Results showed that, with the increase of vitamin D3 contents, both the weight gain rate (WGR) and specific growth rate first increased and then decreased, reaching a maximum value in the 2513 IU/kg group, which was significantly higher than that in the 707 IU/kg group. The feed conversion rate first decreased and then increased and was significantly lower in the 2513 IU/kg group than other groups. Both viscerosomtic index and hepatosomatic index decreased significantly and were significantly higher in the 707 IU/kg group than in other groups. There were no significant differences in the condition factor and survival rate among all groups. The crude fat of whole fish and muscle showed an increasing trend, reaching a maximum value in the 4519 IU/kg group, which was significantly higher than 707 IU/kg group. The crude muscle protein first decreased and then increased; the 8671 IU/kg group was significantly higher than the 1740 IU/kg and 2513 IU/kg groups. The moisture, crude protein, and crude ash of whole fish, as well as the muscle moisture and crude ash, had no significant differences in concentration among all groups. The spine crude ash showed an increasing trend; the 1740~8671 IU/kg groups had significantly higher contents than the 707 IU/kg group. The calcium contents increased with the increase of dietary vitamin D3 levels. Total antioxidant capacity (T-AOC) and superoxide dismutase activities first increased and then decreased, reaching a maximum in the 2513 IU/kg group. The malondialdehyde (MDA) contents first decreased and then increased; the 2513 IU/kg group had significantly lower content than the 707 IU/kg group. Complement 3 (C3) contents first increased and then decreased; the 707 IU/kg group had significantly lower content than the other groups. Alanine aminotransferase and aspartate aminotransferase first decreased and then increased, reaching minimum values in the 2513 IU/kg group. The highest cholesterol and triglyceride values were observed in the 4519 IU/kg group. The calcium (Ca2+) contents first increased with the increase of dietary vitamin D3 levels, with the 707 IU/kg group showing significantly lower content than other groups. The phosphorus and complement 4 (C4) contents were not affected by dietary vitamin D3. After the challenge, T-AOC, MDA, C3, and C4 were significantly elevated; T-AOC, C3, and C4 in the serum had maximum values in the 2513 IU/kg group, and MDA reached its minimum value in the 2513 IU/kg group. With the increase of dietary vitamin D3 content, HSP70, MYD88, IRAK4, TRAF6, and TLR2 in the liver, and head kidney increased first and then decreased before and after the challenge. Gene expression was significantly elevated after the challenge. Taking WGR as the evaluation index, a broken line regression analysis showed that the optimum dietary vitamin D3 requirement was 2 223.45 IU/kg diet for juvenile rockfish S. schlegelii with the body weight of (20.95±0.05) g.

Abstract:With the increasing demand for marine fish and cold-water predatory fish, to ensure its scale and industrialization in the process of breeding and to provide safe, high-quality, and healthy aquatic animal food for the society, the quality requirements of aquatic compound feed in the industry are increasing. Among the three nutrients, predatory fish have a poor ability to use sugar. Protein is the most expensive raw material, and the final product of its metabolism is ammonia, which can lead to the deterioration of water quality. Fat provides energy for fish growth, and essential fatty acids promotes the absorption of fat-soluble vitamins, and promotes protein deposition and utilization as a non-protein energy substance. Therefore, increasing the oil content in predatory fish feed and reducing the use of protein as energy can save feed protein and increase economic benefit. Different fish have different responses to nutrients and energy in feed. The fat metabolism of cultured fish has a certain species-specificity. It is generally believed that cold-water fish have a higher dietary fat requirement. The fat requirement of juvenile salmonids is 20%~30%, much higher than that of warm water fish. As a cold-water fish, Brachymystax lenok has successfully evolved key genotypic or phenotypic traits to adapt to growing at low temperatures, with fat requirements in the range of 17%~19%, slightly below the recommended fat requirements for regular Salmonidae fish. The high-fat feed has been widely used in carnivorous cold-water fish. As physiological conditions limit the demand and utilization capacity of fat, long-term intake of high-fat feed will easily cause fat metabolism disorder and meat quality decline during the breeding period, which seriously affects the health and quality of fish. Nutritional regulation of fat metabolism has become feasible means to reduce body fat deposition and improve meat quality. Therefore, it is particularly urgent to elucidate the fat metabolism mechanism and nutrition regulation of predatory fish. Lipase plays an essential role in lipid metabolism. As an enzyme with affinity at the oil-water interface, glycerol and fatty acids obtained after lipid hydrolysis can provide energy for the animal body and be utilized for its growth. Therefore, adding lipase in feed to regulate body fat nutrition has garnered considerable attention. Exogenous lipase has been well used in broilers and pigs to improve growth performance and physiological metabolism. It is also widely used in fish. However, it is rarely reported in the studies on cold water and freshwater fish nutrition. B. lenok is a rare and cold-water fish found in clear rivers and streams in China. The optimum temperature range for its growth is 18~20℃, and it has very high economic, edible, and research value. This study aims to, through adding different levels of lipase in different fat feed, use lipase in fat metabolism under the effect of high-fat feedstuff stress B. lenok to fat metabolism regulation, nutrition research on B. lenok growth performance, serum biochemical indices, and liver antioxidant effect, for lipase in B. lenok feeds for young fish provide a reference for the application. In this experiment, a 2×3 two-factor experimental design was used to prepare six experimental diets with two lipid levels (180 and 220 g/kg) and three lipase levels (0, 2500, and 5000 U/kg): C-0, C-2500, C-5000 and H-0, H-2500, H-5000. A total of 270 B. lenok with an initial body weight of (7.34±0.16) g were randomly divided into six groups with three replicates and 15 fish per replicate. Fish in each group were fed six different experimental diets for 63 days. The results show that different fat content and lipase level had extremely significant interaction on average body weight (P<0.01) and significant interaction on weight gain rate and specific growth rate (P<0.05). Both fat and lipase had an impact on the growth performance of the body. The body weight, weight gain rate, and specific growth rate of fish in the C-5000 group were the highest. Serum alanine aminotransferase (ALT) levels in H-0, H-2500, and H-5000 groups were lower than those in C-0, C-2500, and C-5000 groups, respectively. There were significant differences between H-0 and H-5000 groups and those in C-0 and C-5000 groups (P<0.05). The low-density lipoprotein cholesterol (LDL-C) in H-0, H-2500, and H-5000 groups was higher than in C-0, C-2500, and C-5000 groups, respectively. There was a significant difference between H-0 and H-2500 groups, and the C-0 and C-2500 groups (P<0.05). With the increase of lipase level, liver glutathione peroxidase (GPX) level increased. The GPX of the 220 g/kg group with the same lipase level was higher than that of the 180 g/kg group. In conclusion, the growth and antioxidant performance of juvenile B. lenok can be significantly improved at a lipid level of 183.7 g/kg, and the lipase level of 5000 U/kg.

Abstract:Litopenaeus vannamei is one of the most important species of cultured shrimp in China. With the continuous development of intensive culture and increase in stocking density and feed feeding, the yield of cultured shrimp and utilization rate of the cultured environment have improved rapidly. However, a large number of residual baits, feces, fertilizers, and drugs have worsened the aquaculture water environment and led to prominent adverse environmental effects, widespread aquatic animal diseases, and severe environmental pollution. Therefore, the development of pollution-free ecological aquaculture, the promotion of industrialized circulating water aquaculture, and the research of efficient and sustainable biological purification systems have become the focus of aquaculture model research. Circulating water aquaculture is a new aquaculture mode in which water is treated and reused using modern techniques. To explore aquaculture water quality and the composition structure of microbial flora in an industrialized circulating water aquaculture system of L. vannamei, the water quality indexes of biological purification aquaculture systems such as primary moving bed biological purification, secondary fixed-bed biological purification, and aquaculture water, and the microbial flora of water and shrimp intestines were measured using high-throughput sequencing technology and bioinformatics analysis. The experimental results showed that the mass concentrations of ammonia nitrogen (NH4+-N) and nitrite nitrogen (NO2–-N) in the culture water met the standards for the healthy culture of L. vannamei after treatment with the biological purification culture system. Concentrations are 0.85 and 0.21 mg/L. Results showed that all water and carrier samples contained a certain abundance of nitrifying Spirillum bacteria, especially on the biological purification carrier, which was significantly higher than the proportion of the bacterial community in water samples. The Chao1 and Shannon indices of chemical fiber filament biological carriers were significantly higher than those in shrimp intestines. Results also showed that the number of microbial species and microbial community diversity on the biological carrier of chemical fiber filaments were higher than those in the intestine of shrimp. The bacterial community composition was characterized at the phylum and genus levels. A total of 46 bacterial phyla were identified in the samples. Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant phyla in the water, and Proteobacteria, Bacteroidetes, Actinobacteria, and Planctomycetes were the dominant phyla in the biological carrier; Proteobacteria, Bacteroidetes, and Firmicutes were the dominant phyla in the shrimp intestine. The abundance of Planctomycetes in the biological carriers increased. The abundance of Proteobacteria and Firmicutes in shrimp intestines was higher than that in water, conducive to the degradation of nitrogen and phosphorus. Bacteroidetes and Firmicutes were the main metabolizing bacteria in the intestine, promoting carbohydrate metabolism and energy acquisition in shrimp. Actinobacteria can degrade organics, including macromolecules such as starch and proteins, and produce antibiotics and other antibacterial substances. The abundance of Actinobacteria in water was very high but decreased in shrimp, which played an important role in the decomposition of organic matter and nitrogen in water. The bacterial species content in the biological purification carrier of the shrimp culture circulating system was lower than that in the water sample, but the microbial diversity was higher than that of aquaculture water. The microorganisms in the biological purification carrier have the characteristics of low abundance and high diversity, possibly due to the increase in the abundance of environment-related functional bacteria on the biological purification carrier and the decrease in some flora species with little impact on the environment, resulting in the aggregation of biofilm bacteria species. In addition, the water and biological purification carrier also contained a certain amount of pathogenic bacteria, such as Vibrio and Mycobacterium. Therefore, it was necessary to regulate the content of pathogenic bacteria in the water environment and shrimp intestine to ensure a healthy shrimp culture. The results showed that the multi-stage biological purification system can effectively increase the microbial flora, promote the metabolism of nitrogen and phosphorus in the water, and regulate the quality of the aquaculture water. The dominant bacterial groups in water, biological purification carriers, and shrimp were different. Proteobacteria and Bacteroidetes were dominant in water biological carriers and intestines, whereas Planctomycetes mainly exist as biological carriers, Firmicutes mainly colonize the intestine, and Actinomycetes mainly exist in water. In this study, high-throughput sequencing technology was used to study the composition of the microbial community structure in the circulating water culture system of L. vannamei. Results can reveal the diversity of its internal microbial community, provide a basis for further revealing the "black box" of biofilters, and have important guiding significance for the study of the construction and denitrification efficiency of biofilters in seawater circulating water culture.

Abstract:Apoptosis is programmed cell death and is regulated by a series of related genes. It is of great significance in resistance to pathogen invasion and maintaining homeostasis in the environment. The release of cytochrome c (Cytc) from the mitochondria into the cytoplasm is a key step in the initiation of apoptosis. Increasing evidence from investigation of Cytc in cell apoptosis and immunity shows that it can participate in cell apoptosis induced by virus infection. For example, white spot syndrome virus (WSSV) stimulation can induce Cytc gene expression in Litopenaeus vannamei hepatopancreas and hemocytes, and the apoptosis of Epinephelus akaara hepatocytes induced by red-spotted grouper nervous necrosis virus (RGNNV) is related to the release of Cytc. However, the role of Cytc-mediated apoptosis in Procambarus clarkii WSSV infection has not yet been reported. Therefore, in this study, the full length of the the cytochrome c gene of P. clarkii (PcCytc) was cloned, and the role of PcCytc in P. clarkii was analyzed. Its expression in various tissues of P. clarkii proved that WSSV infection can induce the expression of PcCytc. The mechanism of PcCytc involvement in cell apoptosis during WSSV infection was also explored using RNA interference technology, to gain a deeper understanding of the potential role of apoptosis-related factors in the immune response of P. clarkii. In this study, PcCytc was cloned using RACE technology, with a total length of 897 bp, including the 163 bp 5′-UTR, 419 bp 3′-UTR, and 315 bp open reading frame; it encoded 104 amino acids. The structure prediction showed that PcCytc contained a conserved Cytochrom_C domain, proving that it is related to energy production and tends to be conserved in evolution. The results of the quantitative PCR showed that the PcCytc gene was expressed in all tissues of P. clarkii. The expression was lowest in the stomach and higher in the gills, intestines, and muscles, which showed, respectively, 9.46, 8.65 and 7.88 times greater PcCytc expression than that in the stomach. PcCytc showed relatively high expression in tissues with high energy consumption, such as the intestines and muscles, which is consistent with previous studies in Penaeus vannamei. The highest expression level was observed in the gills of the main immune and respiratory tissues of P. clarkii, indicating that PcCytc may be involved in the related biological processes. Based on the above results, we speculate that PcCytc may play different functions in different tissues. WSSV infection experiments showed that the expression level of PcCytc in the tested hepatopancreas, intestines, and muscle tissues increased after virus infection, and reached the highest value at 24 h (P<0.01), after which it began to decrease until it returned to a normal level at 96 h; the overall performance was an induced expression pattern. This showed that PcCytc is involved in the process of WSSV infection. In addition, considering that PcCytc can participate in ATP production as a key element in the mitochondrial respiratory chain, the low expression of PcCytc leads to energy deficiency. We speculate that once the virus disrupts the energy metabolism of the host cell, the host may compensate for the loss by upregulating the expression of PcCytc. RNAi technology revealed the role of PcCytc in the process of WSSV infection. At 24 and 48 h after WSSV infection, the WSSV copies of the PcCytc RNAi group were significantly increased compared to the uninterrupted group (P<0.01), and at 72 h were still significantly increased (P<0.05). These results indicate that PcCytc plays an important role in inhibiting the replication of WSSV in P. clarkii and delays the infection process. To further confirm whether PcCytc mainly inhibits WSSV infection through the apoptotic pathway, we tested the expression changes of some important apoptosis-related genes (bcl-2, bax, and caspase-3). Among them, caspase-3 is an effector protein that regulates cell apoptosis, and its expression directly reflects the result of cell apoptosis. The ratio of bcl-2/bax is considered to be an indicator of the process of cell apoptosis; an increase in the ratio indicates that apoptosis has been affected. Inhibition (a decrease in the ratio) indicates that apoptosis was promoted. The test results were as follows: compared with the PBS group, the expression of bcl-2, bax, and caspase-3 genes of P. clarkii in the WSSV group was up-regulated to varying degrees, with a very significant difference in values (P<0.01). This shows that WSSV can cause hemolymph apoptosis in P. clarkii, which is consistent with observations in mud crab and shrimp. In addition, the expression of caspase-3 in the dsCytc injection group was significantly downregulated (P<0.01), indicating that apoptosis was inhibited after interfering with PcCytc. The value of bcl-2/bax in the dsCytc injection group was significantly increased (P<0.01), which supported this conclusion. In summary, our results indicate that PcCytc can inhibit WSSV infection by regulating the apoptotic pathway. The results of this study provide new insights into the immune response of P. clarkii to WSSV infection.

Abstract:Cellulases are a class of modular enzymes with a catalytic glycoside hydrolase (GH) module that hydrolyzes the β-1,4-glucosidic bond of the cellulose chain. Cellulases had been broadly divided into three types: endo-β-1,4-glucanases, exo-β-1,4-glucanases and β-glucosidases, which of synergistic effect lead to the complete degradation of cellulose to glucose. The endo-β-1,4-glucanase is one of the key cellulases in cellulose digestion, which are likely to be the product of a glycosyl hydrolase family 9 (GHF9) gene. In this study, an endogenous cellulase gene, EsGH9-1, was identified from the transcriptome database of the Chinese mitten crab, Eriocheir sinensis, and the sequence of EsGH9-1 was cloned by PCR. The expression levels of EsGH9-1 in different tissues and different developmental stages were studied. In addition, EsGH9-1 mRNA expression and corresponding enzyme activity under different diet conditions were analysed. The result showed that EsGH9-1 genomic DNA (11 679 bp) consists of 15 exons interrupted by 14 introns; EsGH9-1 cDNA (2044 bp) contains an open reading frame of 1740 bp, corresponding to a polypeptide of 580 amino acid residues, with a typical catalytic domain of the glycosyl hydrolase family 9 (GHF9). EsGH9-1 was expressed in various tissues of E. sinensis, particularly high in the hepatopancreas. A significant increase in expression of EsGH9-1 was also observed in megalopa and early-stage larvae. Under different dietary conditions, the relative expression of EsGH9-1 in the plant diet group was significantly up-regulated, which was consistent with the trend of β-1,4-endoglucanase activity. The finding of the endogenous cellulase gene (EsGH9-1) in E. sinensis implies that EsGH9-1 may be involved in the decomposition of plant diet. Furthermore, this work is a fundamental step toward understanding the role of endogenous cellulase in the digestion mechanism of aquatic crustaceans.

Abstract:Pacific abalone Haliotis discus hannai is an economically important aquaculture species in China, whose production accounts for approximately 93% of world abalone aquaculture production. Its weaning phase is a vulnerable life stage associated with high mortality, which has seriously impeded the sustainable development of Chinese abalone aquaculture. Previous research has shown that the intestinal microflora in farmed abalone is affected by numerous abiotic and biotic factors. The weaning post-settlement of diatom-fed abalone on artificial feed may alter the natural succession of microflora in their guts. To study the succession of the intestinal microbiota in the weaning of Pacific abalone, we collected Pacific abalone at the weaning period days 0 (T0), 4 (T4), 10 (T10), 35 (T35), and 40 (T40) by 16S rRNA high-throughput sequencing. Results showed no significant differences in alpha diversity (Shannon, Simpson, ACE, and Chao1) between T0, T4, and T10 and between T35 and T40. The ACE and Chao1 indices tended to decrease with sampling time. Both Good’s coverage values exceeded 99.50%, indicating that the sequence libraries covered most of the microbial community in these samples. A total of 3609 OTUs were identified across all samples after pre-processing, and the unique OTUs tended to decline in the weaning phase, varying from 419 OTUs in T4 to 169 OTUs in T40. The beta diversity of intestinal microbiota showed that the T0/T4 and T35/T40 samples were clustered separately in principal coordinate analysis, with overlaps between T0 and T4, T35 and T40; T10 was in the transitional stage of intestinal microflora succession during the weaning period. In terms of the composition and structure at the phylum level, the dominant bacterial groups in diatom-fed abalone and weaning abalone were relatively consistent, including Proteobacteria, Bacteroidetes, and Firmicutes. The ratio of Proteobacteria decreased, while that of Bacteroidetes increased with the time of weaning. The composition of the dominant genera during the diatom feeding and weaning stages differed significantly at the genus level. The dominant genera of the diatom-feeding stage (T0) included Lentilitoribacter (14.18%), Dinoroseobacter (9.90%), and Neptuniibacter (9.86%). During the weaning stage, Lentilitoribacter (19.70%), Pseudoalteromonas (9.86%), and Arcobacter (5.52%) were the dominant genera in the T4 group; Lentilitoribacter (6.95%), Arcobacter (8.15%), and Vibrio (7.50%) were the dominant genera in the T10 group; and Formosa and Vibrio were the most dominant genera in the T35 and T40 groups. To study the impact of diet change on the microbial communities of the weaning abalone, linear discriminant analysis (LDA) was used to analyze differences in taxon composition among the five sampling groups. A total of 59 biomarkers were identified (LDA>4.0, P<0.05). From T0 to T40, 9, 11, 17, 9, and 13 biomarkers were found, revealing that the dominant species of the intestinal microflora varied significantly over time. The intestinal microflora co-occurrence networks based on robust and significant correlations were constructed to explore synergetic relationships in the samples from T0, T4, T35, and T40. The values of the network topological characteristics, including node, edge, average degree, clustering coefficient, average path length, and modularity, were distinct at different sampling stages. The values at T0 and T35, except modularity, were higher than those in the other groups, indicating that microbial interaction may be more intensive in the T0 and T35 groups. The co-occurrence (positive) and co-exclusion (negative) patterns of microbial genera were distinct between these four groups, especially between T0 and T35/T40. Tax4Fun function predictions showed that the genes encoded by the intestinal microflora of T0 and T4 were mostly related to diseases and cell processing, while T10, T35, and T40 were mostly related to metabolic functions, indicating that the intestinal microbes were involved in various molecular metabolisms, assisting juvenile abalone in adapting to the nutrient-rich artificial feed. This study revealed the adaptation mechanism of H. discus hannai to diet change during the weaning stage from a microbiology perspective for the first time, laying a theoretical foundation for healthy abalone breeding.

Abstract:Spilled petroleum pollution caused by seaborne oil transportation is a major marine environmental problem in the world. Petroleum pollutants contain significant amounts of low-molecular-weight alkanes and aromatics that induce toxic effects once taken up by marine organisms. Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds with the most significant toxic environmental effects. As bivalves, which have a strong enrichment and tolerance capacity for PAHs in water, are widely distributed and easy to obtain, they are often used as model organisms for monitoring and evaluating offshore marine pollution. The blood clam (Tegillarca granosa), a bivalve with high economic value, is widely distributed in mudflats along the coast of Zhejiang Province. Blood clams have characteristics of benthic life, making them more likely to be exposed to petroleum pollutants. At present, there are few studies on the toxic effects of PAH exposure on blood clams. Herein, we chose benzo[a]pyrene (BaP), a typical PAH congener, as a contaminant, and investigated the toxic effects of acute BaP exposure on blood clams and their potential coping mechanisms. Blood clams required for the experiment were collected from Dongji Island and acclimated for a week in the laboratory. After acclimation, several healthy blood clams were randomly divided into the artificial sea water (ASW) (control group), dimethyl sulfoxide (DMSO) (solvent control group, 0.01% VDMSO/VASW), 10 μg/L BaP exposure group, and 100 μg/L BaP exposure groups based on the previous studies of our research group combined with the literature. Each group was set up with three replicates, each containing 40 individuals. The experiment lasted 96 h, and separate glass tanks were used to place all individuals in each replicate. One blood clam was randomly selected from each replicate of each concentration group at 0, 24, 48, and 96 h of exposure. After dissection on ice, the digestive gland was immediately separated using sterile forceps and scissors and stored at –80℃. The paraffin section was then used to observe the lesions of the digestive gland. A colorimetric assay was used to determine the activities of antioxidant enzymes and key enzymes involved in neurotransmission at different time points. The cell damage degree was also determined by measuring the levels of 8-hydroxy-2′- deoxyguanosine (8-OHdG) and malondialdehyde (MDA). In addition, enzyme-linked immunosorbent assays were used to determine DNA methylation levels. Finally, we measured the relative mRNA expression of antioxidant enzymes and performed a correlation analysis. Results showed prominent hemocyte infiltration and necrotic areas in the digestive gland of the blood clam after a total of 96 h of exposure to 10 and 100 μg/L BaP, indicating inflammation. The sloughing of digestive cells from the inner wall of the digestive tubule led to atrophy. Increased oxidative stress was indicated by elevated MDA and 8-OHdG content, leading to damage at the cellular level, such as lipid peroxidation and oxidative DNA damage. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione-S-transferase (GST), increased significantly 24 h post-exposure. This phenomenon further indicated that BaP exposure induced oxidative stress in the blood clam, with the antioxidant defense system actively regulating BaP-induced oxidative stress. The activities of two key neurotransmitter enzymes, acetylcholinesterase and acetylcholine transferase, were significantly reduced, indicating that the stress caused by BaP may induce neurotoxicity in the blood clam. In addition, we analyzed the changes in DNA methylation levels and gene expression of antioxidant enzymes in the blood clam under acute BaP exposure, finding that the DNA methylation levels were significantly decreased compared to pre-exposure, while the mRNA expression of SOD, CAT, and GST was significantly increased. Correlation analysis showed a negative correlation between DNA methylation and the gene expression of antioxidant enzymes, implying that blood clams may activate the antioxidant systems to fight against BaP toxicity by reducing DNA methylation levels. In conclusion, acute BaP exposure exerts a significant toxic effect on the blood clam, primarily characterized by histological damage, oxidative stress, and neurotoxicity. Moreover, changes in DNA methylation levels in the blood clam may be involved in the regulatory process of BaP toxic effects. This study is expected to provide new ideas for the in-depth exploration of the intrinsic regulatory mechanisms of bivalves in response to petroleum pollutant stress, and will be beneficial for resource conservation of the blood clam under the threat of petroleum pollution.

Abstract:Paralytic shellfish toxins (PSTs) are phycotoxins widely distributed worldwide and pose serious marine ecosystems and human health threats. In China's Yellow Sea and the Bohai Sea, Alexandrium spp. has been certified as the major causative dinoflagellate of PSTs, especially in Qinhuangdao, Hebei Province where several poisoning events have been reported, with tens of consumers suffering and some even dying. In terms of these events, mussels contaminated with PSTs were the major cause of consumer poisoning. Therefore, it is vital to reveal the risk of PSTs in these shellfish, which requires scientific opinions on the formation of terminal components of PSTs in mussels. Generally, mussels are not sensitive to PSTs, resulting in the high accumulation of PSTs in their tissues. The PSTs distribute, bio-transfer, and metabolism, and the terminal metabolites pose a risk to consumers. From 2016 to the present, several serious events have occurred in Qinhuangdao caused by PSTs contamination in mussels, which resulted in poisoning by tens of consumers and a huge loss of regional economy. This study exposed purple mussel Mytilus galloprovincialis, the key cultured species of bivalves in Qinhuangdao, to Alexandrium catenella (GY-H25), the predominant producer of PSTs in this area. The accumulation and biotransformation process of PSTs in visceral mass and edible tissue and the accumulation metabolism kinetics were analyzed under exposure to different cell densities by liquid chromatography-tandem mass spectrometry. Our results showed that the growth and toxin production of GY-H25 was stable. The main components of PSTs were N-sulfocarbamoylgonyautoxin (C1 and C2), with the highest algal cell density of 3.5×107 cells/L and the highest production capacity of 2.96 pg STXeq/cell. Toxin-producing algae with the highest production of PSTs per unit volume and algal cell density (22 days) were selected for the exposure experiment. After exposure, the mussel accumulated a high content of PSTs in both visceral mass and edible tissue, with a similar trend in both exposure densities. The whole exposure experiment could be divided into four periods: 0~2 days as the initial exposure period (period Ⅰ); 2~7 days as the period of rapid accumulation (period Ⅱ); 7~12 days as the period of rapid metabolism (period Ⅲ); 12~30 days as the stable period (period Ⅳ). However, the level of PSTs in the visceral mass of mussels in both exposure groups exceeded the maximum residue limit (MRL) of EU (800 μg STX EQ/kg) at the end of the experiment, while that of PSTs in edible flesh was below the MRL. Comparatively, the highest concentration of PSTs in the visceral mass reached 6815.36 μg/kg in the high exposure group, which was 2.61 times that of the low exposure group, with an average accumulation rate of 17.89%, which was significantly higher than that of the low exposure group (13.06%). The results showed that the accumulation of PSTs in the visceral mass and edible tissue of Mytilus galloprovincialis was harmful to its tissues and organs. The toxin excretion rates of the high-and low-concentration groups of PSTs were 74.39 % and 59.15 %, respectively, after 23 days. The average daily elimination rate was 14.4 %, the metabolism rate of Mytilus edulis slowed down in the stable period (period Ⅳ), and some toxins remained in the visceral mass after 23 days of metabolism, which easily formed long-term toxin retention and threatened human life safety. In addition, PSTs in Mytilus galloprovincialis showed a strong biotransformation ability, mainly occurring among C1, C2, and GTX5. According to this research, the transformation pathway from C2 to GTX5 was the main pathway for GTX5 formation during rapid metabolism and stable periods. At the same time, the transformation of C1 to GTX5 was higher than that of C2 to C1, leading to a reduction in the overall proportion of C1. According to the comprehensive evaluation of the metabolic products and toxic equivalence factor (TEF) of GST components in mussels, the metabolic transformation of PSTs in mussels will further promote the formation and proportion of highly toxic GTX5, which will increase the terminal toxicity of PSTs in mussels and may also pose a higher risk to consumers. In this study, compared with the natural conditions, the concentration of toxic algae was far below the harmful algal bloom exposure. The total accumulation of toxins was far lower than the total accumulation of toxins in mussels exposed to harmful algal blooms. Even so, the total amount of residual toxins in the mussel viscera of the high concentration group was still enough to threaten human life safety at the end of the experiment. It was found that the toxin content in the visceral tissue tended to be stable over 12 to 30 days without an obvious downward trend. Even the toxin content increases due to the toxin transformation phenomenon, which means the difficulty in predicting the time required for PSTs in the visceral mass reduced to below the MRLs. Therefore, this study contributes to the scientific assessment of PSTs risk in mussels and provides a basis for establishing regional PSTs monitoring programs.

Abstract:The greenfin horse-faced filefish (Thamnaconus septentrionalis) and rockfish (Sebastes schlegeli) occupy important positions in the offshore net fishery in Shandong Province. Interest in their mariculture has been developing rapidly in recent years as candidates for submerged cage open-sea aquaculture. With the development of breeding techniques and the expansion of large- scale farming, fish disease may become a serious constraint that limits sustainable aquaculture and leads to great economic losses. Epidemiological investigation is the basis of disease control and should be carried out throughout the culture process. In this study, we describe the diseases of T. septentrionalis and S. schlegeli caused by Aeromonas salmonicida subsp. masoucida. In November 2018, an outbreak of T. septentrionalis disease was observed in a farm located in Penglai, Shandong Province, and an outbreak of S. schlegeli disease occurred in the same farm in April 2019, with daily mortalities of 0.4%~1% and about 1%, respectively. The main symptoms in the diseased fish were ulcers, redness, swelling, and bleeding in the mouth. Most diseased fish in the ponds showed “red mouth”. No parasites were observed by the naked eye or light microscope. From the liver, spleen, and kidney of all the diseased fish, many homogeneous colonies were observed after three days incubation on TSA and 2216E agar plates. All strains had the same shape, color, and size, and the 16S rRNA genes of all strains were the same, with high identity with A. salmonicida. The virulence of the isolates was tested experimentally via injection with T. septentrionalis (infected by 2018TS-1) and S. schlegeli (infected by 2019SS-1) in the laboratory to calculate the median lethal dose (LD50). The results showed that the LD50 of 2018TS-1 to T. septentrionalis was 1.78×105 CFU/fish, and that of 2019SS-1 to S. schlegeli was 0.89×105 CFU/fish. The dead fish of the experimentally infected group showed ulcers and red mouth, the same symptoms as in naturally infected fish. Dominant colonies isolated from experimentally infected fish were all identified as A. salmonicida by 16S rRNA gene sequencing, which indicated that 2018TS-1 and 2019SS-1 were the pathogens of T. septentrionalis and S. schlegeli, respectively. Bacterial identification was carried out by 16S rRNA gene analysis and Biolog Gen Ⅲ characterization. The 16S rRNA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: OK258319 and OK258320) isolated from T. septentrionalis and S. schlegeli were analyzed using MEGA5, and the phylogenetic tree derived from 16S rRNA gene sequences clustered the isolates with A. salmonicida. Among the Biolog Gen Ⅲ tests, 31 produced positive reactions or weak positive reactions for both strains (Dextrin, D-Maltose, D-Trehalose, D-Cellobiose, Sucrose, β-Methyl-D-Glucoside, D-Salicin, α-D-Glucose, D-Mannose, D-Fructose, D-Mannitol, Glycerol, Gelatin, Glycyl-L-Proline, L-Arginine, D-Gluconic Acid, Methyl Pyruvate, L-Malic Acid, Bromo-Succinic Acid, Tween 40, α-Keto-Butyric Acid, Acetoacetic Acid, Propionic Acid, Acetic Acid, pH 6, 1% NaCl, 1% Sodium Lactate, L-Aspartic Acid, L-Glutamic Acid, L-Histidine, and L-Serine), and two weak positive reactions for 2019SS-1, while the others were negative. According to the Biolog database, both strains were identified as A. salmonicida. Based on the molecular analysis of 16S rRNA genes and Biolog Gen Ⅲ phenotype results, the isolates were identified as A. salmonicida. The vapA gene, which encodes the outer membrane protein (A-layer protein) and causes the auto-aggregation of bacteria, is a conserved gene with some variation region in A. salmonicida. vapA gene typing is an effective and important method for classifying the molecular types and subspecies of this fish. vapA gene typing was also used in this study to identify subspecies of strains isolated from T. septentrionalis and S. schlegeli. The vapA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: OK300094 and OK300095) were analyzed using MEGA5 with type strains obtained from Gene Bank. The phylogenetic tree derived from the vapA gene sequences clustered 2018TS-1 and 2019SS-1 with type strain ATCC 27013, indicating that the strains isolated from T. septentrionalis and S. schlegeli belonged to A. salmonicida subsp. masoucida, similar to the A-layer type Ⅶ strains, which are all from the northeast Asian and Canadian coasts in the Pacific Ocean. Based on the experimental infection, 16S rRNA sequence analysis, Biolog Gen Ⅲ characterization, and vapA gene typing, we confirmed that A. salmonicida subsp. masoucida is the pathogen of T. septentrionalis and S. schlegeli, and the cause of these two diseases on the farm. This is the first report of T. septentrionalis and S. schlegeli infected by A. salmonicida in industrial aquaculture, as well as the first report of a disease of T. septentrionalis in culture. It has been reported that A. salmonicida subsp. masoucida can infect Atlantic salmon (Salmo salar), turbot (Scophthalmus maximus), sablefish (Anoplopoma fimbria), and tongue sole (Cynoglossus semilaevis) cultured in Shandong Province. In this study, we expanded the host list of A. salmonicida subsp. masoucida to include two new species in aquaculture, T. septentrionalis and S. schlegeli, on the same farm, indicating that A. salmonicida subsp. masoucida may translate and adapt to a new host in a short period. Considering the increasing host and economic losses caused by A. salmonicida in fish culture, the prevention of A. salmonicida subsp. masoucida should be an important objective for mariculture in the future.

Abstract:Neoporphyra haitanensis is a macroalgae available in the south coast of China, and it is one of the most widely cultivated seaweeds in China. In recent years, due to global warming, the continuously high temperatures following the White Dew solar term, has led to the decomposition of seedlings of N. haitanensis in Fujian, Zhejiang and other provinces. This has had a huge impact on the coastal N. haitanensis cultivation industry in terms of production and development. Therefore, investigation of the molecular mechanism of high temperature stress response of N. haitanensis and the high-temperature resistance related genes is essential, and the results can also lay a foundation for the breeding of high-temperature resistant varieties of N. haitanensis. A previous study revealed that the basic region Leucine Zipper (bZIP) family transcription factors are one of the largest and most conserved transcription factor families in plants. The family plays an important role in plant response to abiotic stresses, such as high temperature, drought, and osmosis. bZIP transcription factors regulate plant response to abiotic stress by binding to functional genes or regulatory gene promoter cis-elements to activate and induce downstream gene expression. So far, 127, 89, and 216 bZIP transcription factors have been found in Arabidopsis, rice, and maize, respectively. However, previous studies on bZIP have mainly focused on model plants and only some field crops, and the functions of bZIP in macroalgae have not been reported. To this end, NhbZIP1 was screened based on the whole genome and transcriptomic data of N. haitanensis, and the NhbZIP1 gene was cloned and functionally analyzed by molecular biology and bioinformatics techniques. Its structure and expression pattern were also analyzed. Finally, the NhbZIP1 gene was transformed into Chlamydomonas reinhardtii by the “glass bead transformation” method for gene function verification. In this study, a gene product with a length of approximately 1000 bp was obtained by PCR amplification. After sequencing and BLAST analysis, the gene was identified as the bZIP gene of N. haitanensis and named NhbZIP1. Studies have shown that the open reading frame of NhbZIP1 gene is 825 bp in length and encodes 274 amino acids. There are five low-complexity domains and one BRLZ (115~179 aa) structure. BRLZ is a conserved domain of bZIP family and contains a α-coiled helix structure (121~171 aa). The molecular formula of NhbZIP1 is C1193H1935N339O375S7, and the predicted molecular weight of NhbZIP1 is 27 251.95 Da; its theoretical isoelectric point is 5.03, and it contains 32 negative charge residues and 26 positive charge residues in total. Ala (A) content of the protein was 27%, and Arg (R) content was 4.7%. The total average hydrophilic coefficient was 0.089, which indicates that the protein is hydrophilic, and the instability coefficient was 42.68, which indicates that the protein is unstable. There were 15 potential phosphorylation sites and 12 potential O-linkage glycosylation sites in this protein, which had no signal peptide or transmembrane structure. The protein was located in the nucleus and its characteristics were consistent with those of the genes encoding transcription factors, indicating that NhbZIP1 was a bZIP family transcription factor. Phylogenetic analysis showed that NhbZIP1 gene was isolated from Porphyra umbilicalis and was different from that higher plants, indicating that NhbZIP1 gene was relatively conserved in Porphyra and was genetically distant from higher plants. It is speculated that the NhbZIP1 gene of N. haitanensis has a different evolutionary mode from that of other species. Real-time fluorescence quantitative PCR (qRT-PCR) showed that NhbZIP1 gene was significantly induced by high temperature stress, and the expression level of NhbZIP1 gene was about 3.4 times that of the initial level after 6 h of stress. Under long-term high temperature stress, the heat tolerance of N. haitanensis could be enhanced by enhancing the expression of the resistance gene NhbZIP1. To further clarify the molecular function of NhbZIP1 gene, we transformed it into C. reinhardtii for functional verification. The results showed that the expression level of NhbZIP1 gene was relatively stable before 30 min of high temperature treatment, while the gene expression level was significantly increased after 60 min of the treatment, which was 1.8 times that of the initial level. Under subsequent high temperature stress, the gene expression level remained high, which was about 2.6 times that of the initial level after 180 min of high temperature treatment. The biomass of transgenic lines under heat stress was always higher than that of the wild type, and the difference became more significant with the increase of treatment time. The expression levels of heat shock protein family and genes related to antioxidant system in transgenic lines were significantly higher than that of the wild type. The results showed that NhbZIP1 gene plays an important role in activating the expression of downstream stress-resistant genes in the response to heat stress in N. haitanensis, suggesting that NhbZIP1 gene may enhance the heat tolerance of algae by regulating the expression of HSPs and activating the expression of genes encoding antioxidant enzymes. This study helps to clarify the molecular mechanism of bZIP transcription factor in regulating the response of N. haitanensis to high temperature stress and provides theoretical basis for guiding the breeding of new varieties with high temperature tolerance.

Abstract:With the continuous improvement of living standards and expansion of oyster consumer groups, people are pursuing high-quality oysters, especially with fresh and sweet taste traits. At present, almost all varieties of oysters with different flavors entering the upscale oyster market in China are imported, and their prices are high. Before 2019, among the new oyster varieties approved in China, the dominant traits of most varieties were reflected in growth, shell color, etc. The oyster farming industry has also primarily focused on quantity but not quality, and the contradiction between scale and quality has become increasingly prominent. It can be predicted that with the development of the shellfish industry, the market demand for high shellfish quality will be more and diversified. In addition to pursing traditional traits such as growth rate, the demand for high-quality oysters, such as those with high glycogen, will be more urgent. Glycogen directly affects oysters´ flavor and nutritional quality and is often used as an important criterion to evaluate oyster quality. The efficient, rapid, and high-throughput method for determining glycogen content can provide technical support for cultivating new shellfish species with a high glycogen content. At present, the detection methods for glycogen content are mainly traditional chemical detection techniques and kits. Although these methods have been well developed, they are time-consuming and costly, producing a significant amount of chemical waste liquid. Therefore, they are not suitable for rapid and batch determination of glycogen content in large quantities. As a common modern, fast, and efficient analysis technology, near-infrared (NIR) technology can record the frequency doubling and frequency absorption of the main hydrogen-containing groups using an NIR spectrometer. NIR technology can determine large quantities of samples and has the advantages of being fast and efficient, time-saving, and labor-saving, with no chemical waste liquid generation in the experimental process, offering green environmental protection. After testing by NIR technology, the chemical properties of the samples do not change and do not require significant sample amounts, which can be used for subsequent recycling. The NIR scanning samples used in this experiment can be quickly recovered and stored in a refrigerator after obtaining spectral data through a short period of NIR scanning without affecting the use of subsequent samples. In general, the NIR technique has many advantages in determining glycogen content, and are applicable for improving oyster quality traits. This method has many advantages, such as a wide application range, and is fast, efficient, convenient, and accurate; it has been widely applied in aquatic science research. This is especially true in quality element detection research. NIR quantitative models of water, fat, glycogen, total protein, amino acid, taurine, zinc, selenium, and calcium in Crassostrea glomerata, C. angulate, Saccostrea glomerata, and C. virginica were established, with demonstrably high accuracy. Among them, NIR analysis technology has been successfully applied to breeding a new Crassostrea gigas species “Lu Yi 1,” significantly improving oyster breeding efficiency. Therefore, NIR assays can effectively overcome problems of chemical detection methods, which are time-consuming, laborious, and expensive; this is highly significant for breeding oysters with high-quality traits. The NIR spectroscopy model can be used to quickly and accurately predict glycogen content. Using C. ariakensis as the research object, the glycogen content of 909 samples in seven tissues including mantle, gill, adductor muscle, hepatopancreas, labial palps, gonad, and most soft oyster parts were determined by the micro-reaction system and method of oyster glycogen content. The corresponding spectral data were obtained using a Fourier NIR spectrometer. The spectral data and glycogen content data were analyzed and processed using TQ Analyst software, and NIR quantitative models of six tissues and all the soft parts of oysters near the Jiang River were established, and 1/9 samples of the total sample size were randomly selected for external validation and cross-validation of the models. The results showed that the measured glycogen content ranged from 7.11 to 602.20 mg/g, which had a wide range and was suitable to establish the model. This study aimed to establish a NIR model for the freeze-dried tissues of C. ariakensis to realize the rapid and accurate detection of glycogen content. This study obtained the glycogen content and spectral data of 909 samples using the micro-reaction system method and NIR technology. Combined with the least-squares method, the glycogen content prediction model of six tissues and the whole soft body of C. ariakensis was established and verified. Results also show that the model is optimal after the first derivative, multiplication scattering, and smoothing pretreatment of the measured spectral data. The modeling correlation coefficients (RC) of the seven models ranged from 0.971 6 to 0.996 3, indicating that the predicted values of the seven models were highly correlated with the actual chemical values. The correlation coefficients of external validation (REV) and cross-validation (RCV) were between 0.949 0~ 0.990 8 and 0.969 4~0.996 9, respectively, indicating that the established model could accurately predict the glycogen content of the corresponding tissue samples of C. ariakensis. This method rapidly and accurately determines the glycogen content of oysters and has application value in the field of improvement of oyster characteristics and quality.

Abstract:At present, local and foreign research reports on Thunnus thynnus have focused on fishery biology, fishing, and genetic diversity, among others. There are few reports on nutrition and flavor related to the different muscles of T. thynnus. To scientifically evaluate the nutritional quality and major flavor of different muscle parts of T. thynnus, the basic nutritional components, amino acid composition, fatty acid composition, and volatile flavor substances of dorsal and abdominal muscles were compared. Except for ash, the moisture, crude protein, and fat contents varied significantly between the dorsal and abdominal muscles (P<0.05). The moisture contents of the dorsal and abdominal muscles were 57.79 g/100 g and 50.16 g/100 g, respectively. The protein content was 24.70 g/100 g and 18.61 g/100 g, respectively. The protein content of dorsal muscles was 24.70 g/100 g, which was 1.33 times that of the abdomen. The crude fat content of the dorsal muscles was 19.34 g/100 g, and in the abdomen was 30.29 g/100 g, which was 1.57 times that in the dorsal muscles. The crude ash content of dorsal and abdominal muscles was 1.01 g/100 g and 1.08 g/100 g, respectively. Seventeen amino acids were detected in different parts of T. thynnus. The total amino acid content differed significantly between the dorsal (22.07±0.74) g/100 gand abdominal muscles (16.57±0.47) g/100 g. The essential amino acid content of dorsal and abdominal muscles was (8.61±0.29) g/100 g and (6.28±0.17) g/100 g, respectively. The content of glutamate was the highest among the amino acids, followed by aspartic acid, and the content of cystine was the lowest. The ratio of essential amino acids to total amino acids of different muscle parts of T. thynnus was 40%, and the ratio of essential amino acids to nonessential amino acids was >60%, which was close to the ideal protein nutrition evaluation model recommended by the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). Thus, the dorsal and abdominal muscles are high-quality proteins. The umami taste of aquatic products is closely related to the content of umami amino acids. Four types of umami amino acids showed the highest content levels, namely glutamic acid, aspartic acid, alanine, and glycine (in descending order of content level). The proportion of umami amino acids in dorsal and abdominal muscles was 34.93% and 35.91%, respectively, both exceeding 30%, which indicated that the dorsal and abdominal muscles of T. thynnus have a delicious quality. In the dorsal and abdominal muscles, 24 and 29 kinds of fatty acids were detected, including 8 and 12 kinds of saturated fatty acids, 6 kinds of monounsaturated fatty acids, and 10 and 11 kinds of polyunsaturated fatty acids, respectively, and there was a significant difference among the contents of different types of fatty acids. The contents of myristic acid, palmitic acid, and stearic acid were higher than those of other saturated fatty acids. The contents of oleic acid and eicosanoic acid were higher in monounsaturated fatty acids. The total content of eicosapentaenoic acid and docosahexaenoic acid in the dorsal and abdominal muscles accounted for 88.55% and 75.13% of polyunsaturated fatty acids, respectively. Therefore, the dorsal and abdominal muscles have good functional properties. The FAO/WHO recommends that the intake ratio of n-6 to n-3 polyunsaturated fatty acids in the diet should be 4:6, and the ratio of dorsal and abdominal muscles ranged from 0.41 to 0.44, which is much smaller than the standard. These results indicate that T. thynnus is rich in n-3 polyunsaturated fatty acids. A total of 39 volatile substances were detected in the dorsal and abdominal muscles of T. thynnus, including aldehydes, ketones, alcohols, esters, acids, alkenes, and compounds containing nitrogen and sulfur. PCA analysis showed that there was a significant difference between the odors of the dorsal and abdominal muscles of T. thynnus. Gas chromatography–ion mobility spectrometry can effectively be used to distinguish the different volatiles associated with the muscles. The relative contents of 2-methylthiophene, (E)-2-hexen-1-ol, pentanoic acid, alpha-pinene, propanoic acid, butyl butanoate, 2-pentylfuran, pentyl butanoate, dipropyl disulfide, diallyl disulfide, ethylheptanoate, alpha-terpineol, methylisobutylketone, 2-methylbutanal-D, and 2-methylbutanal-M were higher in the abdomen; the relative contents of (E)-2-pentenal, N-nitromethylethylamine, methylpyrazine, 3-methylbutanol, 1-2-dimethylbenzene, triethylamine, E-3-hexene-1-ol, 4-methyl-2-pentanol, ethyl 2-methylpropanoate, 2-butanone, ethyl acetate, 3-butenonitrile, and 2,4,5-trimethylthiazole were higher in abdominal muscle. The difference in composition and content caused the dorsal and abdominal muscles to form their own flavor characteristics, which were mainly related to the amino acid and fatty acid composition of the two parts. In general, the dorsal muscles mainly presented a fatty fragrance, whereas the abdominal muscles mainly presented a clear fragrance. The dorsal and abdominal muscles of T. thynnus are rich in nutrients and n-3 polyunsaturated fatty acids. Both muscles have a delicious taste, though they vary in flavor characteristics. This study provides a scientific basis for the deep processing and utilization of T. thynnus.

Abstract:Antarctic krill (Euphausia superba), an important group of marine zooplankton in the Southern Ocean, is the only fishery resource with extremely rich reserves and a low degree of development in the world. Antarctic krill is considered to be the greatest potential source of high-quality marine protein resources due to its abundant biomass and high protein content. Peptides prepared from Antarctic krill exhibit multiple physiological activities, including osteoporosis relief, glucose metabolism regulation, blood pressure amelioration, antioxidation, fatigue alleviation, and anti-aging activity. The production and development of Antarctic krill peptides has recently become an industry focus; however, existing research has been limited to the optimization of enzymatic hydrolysis processes, mainly involving the screening of suitable enzymes and the optimization of enzymatic hydrolysis conditions. Due to the high mineral content of Antarctic krill and the introduction of buffer salt in the process of enzymatic hydrolysis, current Antarctic krill peptides products have a high salt content, which leads to poor sensory experience and health risks. Hence, a process for desalination of Antarctic krill peptides is needed. Desalination methods for bioactive substances include dialysis, ultrafiltration, nanofiltration, electrodialysis, and macroporous resin adsorption. In the field of membrane separation, nanofiltration technology has been widely used in the purification, concentration, and desalination of food components owing to its advantages: low operation cost, no introduction of exogenous substances, no destruction of materials, and low rejection rate of monovalent ions. In order to improve product quality and ensure market expansion, the process of desalination of Antarctic krill peptides using nanofiltration technology was studied and optimized in this study. Defatted Antarctic krill powder was enzymatically hydrolyzed by alkaline protease to obtain Antarctic krill peptides for further use. The main factors affecting the desalination effect of Antarctic krill peptides (peptides concentration, nanofiltration pressure, and cycle times) were optimized by single-factor and orthogonal tests, using the desalination rate and protein loss rate as evaluation indexes. The experimental optimization ranges included peptides concentration of 1%~5%, nanofiltration pressure of 0.6~1.4 MPa and cycle times of 1~5. The salt contents of the samples before and after desalination were quantified using the silver nitrate titration method; the protein contents of the experimental samples were quantified using the Lowry colorimetric method. The quality indexes of the Antarctic krill peptides after treatment (including the basic nutritional composition: moisture content, protein content, ash content, salt content; amino acid composition; and molecular weight distribution) were systematically evaluated by the corresponding national standard methods. All experiments were performed in triplicate, and data were expressed as mean ± standard deviation. Excel 2016, IBM SPSS 20.0, and Origin 2018 were used for data analysis and chart drawing. Single-factor tests revealed that peptides concentration of 3%, nanofiltration pressure of 1.0 MPa and a cycle time of 2 could be selected as the design basis for the L9 (33) orthogonal test. The range value of the orthogonal test indicated that the degree of influence of the three factors on the desalination effect was as follows: peptides concentration > cycle times > nanofiltration pressure. The optimum conditions for desalting Antarctic krill peptides obtained by k value analysis were as follows: peptides concentration of 3.0%, nanofiltration pressure of 1.2 MPa and a cycle time of 3. Under the optimal condition, the desalination rate of the Antarctic krill peptides reached up to (86.35±2.11)%, and the protein loss rate was controlled at (9.10±0.35)%, demonstrating the feasibility of the process. The salt content of the Antarctic krill peptides after desalination was reduced to (1.14±0.12)% and the protein content was (92.73±2.29)%. The molecular weights of the Antarctic krill peptides after desalination were mainly distributed between 189 Da and 6500 Da, of which the proportion of peptides with molecular weight less than 3000 Da was (88.91±2.19)%, conforming to the molecular weight distribution range of bioactive peptides. The amount of essential amino acids in the Antarctic krill peptides after desalination accounted for (40.06±0.10)% of the total amino acids, and the ratio of essential amino acids to nonessential amino acids was (66.82±0.28)%. The amino acid compositions of the Antarctic krill peptides after desalination were ideal and met the standard stipulated by the FAO/WHO. The established nanofiltration desalination process presented good treatment effects, and the obtained peptides were of good quality and high nutritional value. The production of Antarctic krill protein-related products may be the next key development for the processing industry, since the sole high-value products of Antarctic krill at present are Antarctic krill oil and its derivatives. The established nanofiltration desalination process has practical application value and would provide technical support for the development of high-quality Antarctic krill peptides. This research provides scientific support for the efficient utilization of Antarctic krill resources.