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Abstract:In recent years, the fishery resources in China's coastal and inland waters have declined seriously, along with a significant decrease in the number of economic fish stocks in many natural waters. Enhancement and release are important measures to conserve fishery resources, protect biodiversity and replenish the fishery environment. Since the 1980s, enhancement and release have gradually become an important way to increase fishery resources in China. With the yearly increase in variety, quantity, and scale of enhancement and release, the problem with the low efficiency of enhancement and release has become increasingly prominent. Therefore, a scientific and reasonable variant of the enhancement and release technique is the key to ensure its successful role in resource proliferation, including prior scientific planning as well as an accurate evaluation post release. At present, there are many studies on release assessment methods. Ecopath model method and empirical assessment method are often used to carry out pre-release planning research. Ecopath model method determines the release quantity in the water area by calculating the ecological capacity of the release water area. However, this method, like the empirical assessment method has the disadvantage of an extensive assessment of the capacity limiting its accuracy in planning the release quantity. The commonly used methods for evaluating the effect of post-release include external tagging method and statistical evaluation method. The external tagging method causes certain damage to fish and has poor stability, making it unsuitable for large-scale enhancement and releasing. The statistical evaluation method needs regular follow-up post release, making it manpower-, resources- as well as cost-intensive. In order to formulate scientific enhancement and release strategies and improve the effect of breed enhancement and release, the EnhanceFish model was introduced in this study using Acanthopagrus latus as the research object, Nanlang water area as the research area, and considering the life cycle, growth rate, mortality, sexual maturity, heritability, recruitment, economic cost and effort as the study parameters. The enhancement and release model of A. latus in Nanlang water was constructed, and the optimal release size, ecological capacity, population structure, and economic benefits of A. latus were scientifically evaluated in order to provide scientific guidance for the rational and standardized formulation of enhancement and release strategies. In this study, the effects of spawning stock biomass, profit, and fishing effort on the ecological structure of A. latus were comprehensively considered, and an intuitive data model was constructed to determine the range of release quantity and fishing effort. The results showed that the natural recruitment amount increased with the increase in releasing quantity. When the release quantity was 2 million, the natural recruitment amount reached the progressive value of 1.6 million. Therefore, the number of A. latus released in Nanlang water area was 0–1.6 million. While evaluating the relationship between fishing effort and economic benefit before release, the profit was 1 000 CNY assuming the current fishing effort E=1. When the fishing effort was E<1, the profit increased with the increase in fishing effort. When it was E>1, the profit decreased with the increase in fishing effort. When it was E>2, the profit was 2 000 CNY, which has no research value. Therefore, the limit of fishing effort E is 0–2.

Abstract:The Argentinean shortfin squid, Illex argentinus, is an oceanic cephalopod displaying characteristics of short lifespan, fast growth rate, and semelparous reproduction. Here, we aimed to comprehensively understand the squid´s life history strategy by investigating the body condition and gonadosomatic index of mature male squids collected from the high seas of the southwest Atlantic Ocean during the 2018–2021 fishing season. Our evaluations used residual index analysis and mixed-effects models to evaluate the relationships between body condition and gonadosomatic index and the key oceanic variables, including sea surface temperature (SST) and chlorophyll a concentration (Chla). The application of the mixed-effects models allowed us to explore the potential influences of habitat environment on somatic and reproductive growth. Our results showed that mature male I. argentinus had a measured mantle length (ML) between 143 mm and 291 mm and a body weight between 89 g and 559 g. We also noted that the largest individuals were collected in 2020, whereas the smallest were collected in 2018. The b values for individuals from 2018 to 2020, estimated using a power regression of body weight versus mantle length, were significantly different from the assumed isometric growth coefficient b value (equals to 3). The standardized residuals of BW-ML relationships indicated that the individuals collected in 2020 retained the best body condition, whereas the individuals from 2018 to 2021 displayed poor body condition. In contrast, mature male I. argentinus showed the lowest gonadosomatic index in 2020, followed by 2018 and 2021. Mixed-effects model evaluations revealed a significant correlation between body condition, SST, and Chla. The individuals displayed a decreasing trend in body condition in response to increasing sea surface temperature, from 9.0 ℃ to 12.5 ℃, and body conditions improved when the chlorophyll a concentration was around 1 mg/m3. Mixed-effects modeling also revealed that the gonadosomatic index was positively correlated with SST, demonstrating a relatively high value at approximately 15 ℃. Taken together, these findings indicate that there is a trade-off between body condition and reproductive investment, and that SST has a significant influence on body condition and reproductive investment for mature male I. argentinus. Additionally, chlorophyll a concentration also demonstrated a significant influence on the body condition of mature male I. argentines.

Abstract:Fish meal and fish oil are important sources of protein and lipid in feeds. The lack of fish meal and fish oil supply has become an important limiting factor for the aquaculture industry. The proportion of fish oil and fish meal used in commercial feeds for farmed fish and crustaceans is continually decreasing, while the carbohydrate content is increasing. Carbohydrates are one of the three major nutrients that provide energy for the body. They have a much lower production price than protein and fat. Starch is a polymeric carbohydrate made from glucose molecules; it is the most commonly used in aquatic feeds and can replace fish meal and fish oil to reduce feed cost. Therefore, it is important to fully explore the nutritional function of carbohydrates for aquaculture. Hypoxia is a common stress in aquaculture, and acute hypoxia may lead to massive mortality of cultured fish in a short period of time resulting in serious economic losses to aquaculture. Therefore, it is crucial to find ways to improve the acute hypoxia tolerance of fish. Fish mainly use glucose for energy under acute hypoxia. Hypoxia inhibits oxidative phosphorylation in mitochondria and activates the anaerobic glycolysis pathway. Glucose degrades to produce lactate and ATP. Carnivorous fishes have limited absorption and utilization capacities to feed carbohydrates, unlike omnivorous fishes. Dextrin is an intermediate starch hydrolysis product, with a molecular weight between starch and glucose; it has good adhesion properties and is more easily digested and absorbed than starch. Therefore, we hypothesized that the use of easily digestible carbohydrate in the feed is an effective way to improve the acute hypoxia tolerance of fish. Takifugu rubripes is loved by Japanese and Korean consumers owing to its delicious taste and high nutritional value. It is a characteristic species of Chinese mariculture fish. It is mainly cultured in a high-density factory, and the dissolved oxygen in the water often relies on water exchange and an oxygenation pump; its gill cover is degraded and is at risk of acute hypoxia. This study determined the effect of the addition of corn starch or dextrin (corn starch hydrolysate) to the feed on growth performance, acute hypoxia survival rate, metabolite content, and the hypoxia inducible factor (HIF) signaling pathway of T. rubripes after 8 weeks of feeding. There were no significant differences in weight gain, feed conversion ratio, hepatosomatic index, viscerosomatic index, condition factor, and body composition between the starch group and the dextrin group (P>0.05). However, the survival rate during acute hypoxia of the dextrin group was significantly higher than that of the starch group (P<0.05). There were no significant differences in the liver glycogen and lactate contents, and lactate dehydrogenase A4 (ldha) gene expression between the starch group and dextrin group (P>0.05) during normoxia. However, the lactate content and ldha gene expression were significantly higher in the liver of the dextrin group than those in the starch group (P<0.05) during hypoxia. This indicated that feeding dextrin strongly activated anaerobic glycolysis to provide more energy under hypoxia. The serum triglyceride (TG) content significantly increased in the dextrin group compared with the normoxia groups, although the TG content in the serum and liver significantly decreased in the starch group after acute hypoxia (P<0.05). This suggested that feeding starch promoted lipid catabolism and oxygen consumption. There was no significant difference in the total soluble protein content of the muscle between the starch group and the dextrin group (P>0.05) during normoxia. However, the total soluble protein content of the muscle decreased in the dextrin group compared with the starch group (P<0.05) after acute hypoxia. Meanwhile, the total muscle soluble protein content, and the gene expression of liver v-akt murine thymoma viral oncogene homolog 1 (akt1), and mechanistic target of rapamycin kinase (mtor) significantly decreased in the dextrin group after hypoxia compared with the normoxia group. However, the liver mtor gene expression significantly increased in the starch group after hypoxia (P<0.05). This data demonstrated that protein synthesis was inhibited in the dextrin group under hypoxia. Hypoxia inducible factor (HIF) is the most critical transcription factor in cellular response to hypoxic stress. The dextrin group had higher hypoxia inducible factor 1 subunit alpha like (hif-3α) gene expression in the liver and lower hypoxia inducible factor 1 subunit alpha a (hif-1α) gene expression in the muscle (P<0.05) compared with the starch group during normoxia. The gene expression of liver hif-1α and hif-3α and muscle hif-1α and vascular endothelial growth factor A (vegfa) significantly increased in the dextrin group compared with the starch group (P<0.05) during hypoxia. This proved that feeding dextrin strongly activated the HIF signaling pathway under hypoxia. In summary, replacing starch with easily digestible dextrin in the feed did not affect the growth performance. Instead, it activated the HIF signaling pathway and anaerobic glycolysis to provide more energy for fish. Meanwhile, feeding dextrin inhibited lipid catabolism and protein synthesis, and reduced oxygen consumption to improve the acute hypoxia tolerance of T. rubripes. The study provides important guidance for the formulation design of hypoxia-tolerant feed and healthy development of aquaculture.

Abstract:Terrestrial animal protein sources contain less antinutritional factors, high protein content, and functional factors, which are beneficial to fish health. Among them, poultry byproduct meal (containing 65%–73% protein rich in vitamins) and porcine meat meal (containing 45%–60% protein and high contents of proline and glycine) are the most widely used meals in aquatic compound feeds, and are important fish meal replacement sources. As a carnivorous fish species, largemouth bass (Micropterus salmoides) is highly dependent on dietary fish meal, and the level of fish meal added in its commercial feeds is up to 50%. However, the rising price of fish meal increases the farming cost of M. salmoides. Therefore, it is necessary to identify a suitable alternative protein source to reduce the amount of dietary fish meal and the feed cost. Therefore, seven compound feeds (D1–D7) were prepared in this study. The added ratios of fish meal/poultry byproduct meal/porcine meat meal were as follows: 45.0/22.6/0, 37.1/22.6/8.0, 28.8/22.6/16.0, 45.0/14.5/8.0, 45.0/5.3/16.0, 41.6/18.0/8.0, and 37.0/13.8/16.0. Juvenile M. salmoides (initial body weight ~55 g) were fed the above diets for 60 days with five replicates in each group. The effects of the animal protein source combination on the growth performance, tissue biochemical indices, muscle texture characteristics, liver protein metabolism, and intestinal inflammatory factor-related gene expression were evaluated. The water temperature during the feeding trial was 27.4–32.3 ℃ and the ammonia nitrogen concentration was 0.1–0.2 mg/L. After the feeding experiment, three fish were randomly selected from each cage to collect the serum, liver, intestinal tract, muscle, and other samples, which were then stored at –80 ℃. In addition, three fish were randomly selected from each cage to determine their morphological indices. At the same time, two fish were selected from each cage to determine the muscle texture characteristics and the whole fish proximate composition. Physiological and biochemical indices of serum and liver tissues, albumin, urea nitrogen (BUN), total amino acid (T-AA), alanine aminotransferase (ALT), aspartate aminotransferase, total protein (TP), and blood ammonia (SA) levels), were determined using commercial kits, and the texture characteristics of muscle were determined by using a texture analyzer. The moisture, crude fat, crude protein, and ash contents of whole fish and muscle were determined by atmospheric drying, Soxhlet extraction, Kjeldahl nitrogen determination, and Muffle furnace incineration, respectively. Real-time quantitative PCR was used to determine the expression levels of genes related to liver protein metabolism and the intestinal inflammatory response. All test data were expressed as the mean±standard error, and multiple comparisons were made by the Tukey test, with P<0.05 indicating a significant difference. The results showed that, compared with other groups, the final body weight, weight gain rate, and specific growth rate of fish in the D3 group were significantly higher, and the feed conversion ratio was significantly lower (P<0.05). There were no significant differences in the condition factor, hepatosomatic index, viscerosomatic index, and survival rate among all groups (P>0.05). The whole-body crude protein content in the D3 group was significantly higher than that in the D1 group, and the crude lipid level in the D3 group was significantly lower than that in the D6 group (P<0.05). In terms of tissue physiological and biochemical indices, there were no significant differences in the activities of BUN and ALT in serum, SA content, and ALT activity in the liver among all groups (P>0.05). The serum T-AA content of fish in the D3 group was significantly higher than that in the D1 and D4 groups (P < 0.05), but the AST activity in the D3 group was significantly lower than that in the D5 group (P<0.05). The liver TP content in the D3 group was significantly higher than that in the D7 group (P<0.05). There were no significant differences in the serum TP and liver ALT contents in the D3 group compared with those of the other groups (P>0.05). In terms of muscle quality, the muscle hardness, adhesion, and mastication in the D3 group were significantly lower than those in the D4 and D6 groups, respectively (P<0.05). There were no significant differences in the muscle adhesiveness, elasticity, cohesiveness, moisture, crude protein content, crude lipid content, and ash content among all groups (P>0.05). In addition, the mRNA expression levels of intestinal il-10 and liver tor, s6k1, akt, and pi3k in the D3 group were upregulated, and were significantly higher than those in the D7 group (P<0.05). The mRNA expression levels of il-1β and il-6 in the intestines and 4ebp-1 in the liver of the D3 group were significantly lower than those of the D1 group (P<0.05). These results indicated that combined use of 28.8% fish meal, 16.0% porcine meat meal, and 22.6% poultry byproduct meal had the best growth promotion effect on M. salmoides, and was able to improve liver protein synthesis and maintain intestinal health. The results of this study provided technical support for reducing the dependence of M. salmoides compound feed on fish meal.

Abstract:Trachinotus ovatus, commonly known as golden pompano, is a euryhaline warm water carnivorous fish. It has the characteristics of fast growth, simple feeding, delicious meat, strong stress resistance, and high survival rate. It can accept compound feed throughout its growth. It is popular among fish breeders and consumers because of its moderate specifications and affordable price. With an annual output of 240 000 tons, it has become one of the most important marine fish breeding species in the southern coastal areas of China. As a marine carnivorous fish, it has specific requirements relating to the levels and sources of dietary protein and fat, and a strong dependence on fish meal and fish oil, which are limited resources with high prices, which also determines its high feed cost. However, compared with other rare sea fish, its price is low and the profit margin of breeding is low (2–4 CNY/kg), thus, easily leading to the loss of breeding enterprises and individual businesses. Therefore, it is necessary to develop efficient and low-cost compound diets and reduce the supplemental level of fish meal oil in diets to solve the bottleneck problem of golden pompano fish breeding. Previous studies have shown that T. ovatus subjected to a high efficiency and low fish meal diet exhibited excellent growth and health in pond cage culture. To further evaluate the application effect of this feed in deep-sea cage culture, an experimental feed (crude protein 47.66%, crude fat 7.98%) based on the formula feed of a low fish meal diet was produced by a feed company with a large-scale production process (feed production using large machinery and mass production in a feed mill with an hourly output that can reach more than 10 t using equipment such as oil sprayer machines, where the fat source is added by spraying). A commercial feed from a well-known brand was used as the control diet (crude protein 47.75%, crude fat 9.63%). Large-sized golden pompano (mean body weight ~262 g) were provided by Yangjiang Haina Fisheries Limited and kept for 2 weeks at the deep-sea cage breeding base in Dasuo Island, Yangjiang (12–20 m depth, about 15 km offshore) to adapt to the test environment. During the temporary feeding period, a well-known commodity was used for feed. Overall, 150 000 healthy large-sized golden pompano with neat specifications (initial body weight ~260 g) were selected and randomly assigned to six deep-sea cages (HDPE C60 floating cages, circumference 60 m, 25 000 fish per cage). Each feed was provided in three parallel cages for 33 days (April 29 to May 31, 2021). During breeding, full food was provided twice a day (07:00 and 17:00). During the experiment, the seawater temperature was 20.00~29.00 ℃. Dissolved oxygen was 6.30~7.80 mg /L. The results showed that the growth performance of fish was not statistically different between the two groups (P>0.05). However, compared with the control group, the weight gain rate and specific growth rate of fish-fed experimental diets increased by 14.43 % and 8.19 %, respectively, and the average daily weight gain increased by 0.68 g. In terms of muscle nutrition and texture characteristics, the muscle lipid contents of the fish-fed experimental diets were significantly higher than those of fish-fed control diets (P<0.05), but the muscle moisture content significantly decreased (P<0.05). The edible quality and texture characteristics of muscle were comparable between the two groups (P>0.05). Compared with the control group, the serum protein, triglyceride, total cholesterol, and low-density lipoprotein contents, as well as the activity of aspartate aminotransferase, of fish fed the experimental diet were significantly decreased (P<0.05), and the hepatic total cholesterol content of the experimental group was significantly decreased (P<0.05). There was no significant difference in liver antioxidant capacity between the two groups (P>0.05). In addition, the feed cost per 1 kg of fish receiving the experimental diet was 18.80% lower than that of fish receiving the control diets, and its culture benefit was increased by 62.12%. The results showed that the experimental diet (high efficiency and low fish meal diet) not only promoted growth, but also improved the muscle fat level and serum lipid metabolism of the fish. These results indicate that the high efficiency and low fish meal diet can be applied in the culture of golden pompano within deep-sea cages. In this study, a high efficiency and low fish meal diet for T. ovatus was developed by using amino acid balance technology and fatty acid precision nutrition technology in deep-sea cage large-scale culture. Through the analysis of growth performance, serum biochemical parameters, liver lipid metabolism, and antioxidant properties, it was found that the growth promoting effect of test material was comparable to that of commercial material, and could improve the muscle quality and liver health of golden pompano. Use of the experimental diet could also reduce the cost of breeding, improve the economic benefits, and result in high economic value. The results indicate that the experimental high efficiency and low fish meal diet for T. ovatus has a good application effect and excellent market development prospects, and also has important practical guiding significance for the large-scale production and application of high efficiency low fish meal compound feed, solving the problem of aquaculture bottleneck and facilitating deep-sea golden pompano culture.

Abstract:A 9-week feeding trial was performed to investigate the effects of substituting fish meal (FM) with defatted mealworm Tenebrio molitor meal (TM) on the growth, body composition, serum immune index, as well as histology, barrier functions, digestive enzymatic activities, and microbial communities of the intestine of spotted seabass (Lateolabrax maculatus). In this study, the basal diet was formulated to contain 30% FM, and five experimental diets were formulated by replacing FM with TM at different levels: 0 (TM 0), 5% (5% TM), 10% (10% TM), 15% (15% TM), and 20% (20% TM). Juvenile spotted seabass (2.83±0.02) g were randomly assigned to five treatments with three replicates and 20 fish per replicate. The results showed that the weight gain rate, specific growth rate, and protein productive value of spotted seabass first increased and then decreased with an increase in TM. Among the treatments, there were no difference in the feed efficiency, feeding rate, survival, hepatosomatic index, abdominal fat ratio, or body composition (P>0.05), but the viscerosomatic index was higher in the 5% TM treatment than that in the 20% TM treatment (P<0.05). The serum lysozyme activity was induced in the 5% TM treatment compared to that in other TM treatments (P>0.05). Intestinal histomorphology of spotted seabass was altered with increased dietary TM levels. Compared with the FM treatment, the intestinal villus width, villus height, and muscular thickness were increased significantly in the 5% TM treatment, while all three indices were decreased significantly in the 20% TM treatment (P<0.05). Meanwhile, the expression of the pro-inflammatory gene IL-1β was significantly down-regulated in 5%–15% TM treatments compared to that in the FM and 20% TM treatments (P<0.05). A similar pattern was observed in the expression of the anti-inflammatory gene IL-4. The transcripts of genes associated with barrier functions (ZO-1 and Ocln) were significantly up-regulated in the 5% TM treatment (P<0.05). However, the activity of digestive enzymes (protease and lipase) was not different among all treatments (P>0.05). Furthermore, the alteration of intestinal microbial communities was observed with increasing dietary TM levels. Higher genus abundance of Bacillus was observed in the TM treatments compared to that in the FM treatment (P<0.05), and the relative abundance of Plesiomonas tended to decrease in the 5% TM and 10% TM treatments. In conclusion, substituting fish meal with 5% TM can improve the growth and intestinal health of spotted seabass, while 15% TM had no negative effects on fish. However, excess dietary TM (20%) inhibits growth, causes histopathological damage, and alters the composition of intestinal microbial community in L. maculatus. According to the results of the quadratic regression model, the level of fishmeal substitution by TM in the diet of spotted seabass should not be greater than 7.31%.

Abstract:In recent years, with the increase of varieties and the expansion of scale in aquaculture, as well as the rapid development of the intensive and industrial aquaculture industry, the demand for fish meal has increased significantly. On the other hand, due to global warming and environmental pollution, marine resources have reduced and the production of high-quality fish meal is gradually decreasing. The soaring prices of fish meal increase the feed cost in the process of aquaculture, severely decrease the economic benefits of aquaculture farmers, greatly limit the use of fish meal in aquatic feed, and hinder the sustainable development of the aquaculture industry. Therefore, it has become an important research subject in the aquatic feed industry to find new fish meal substitutes and reasonably reduce the amount of fish meal in feed. Crickets have high nutrient concentrations (55%–73% crude protein, high unsaturated fatty acid levels, and sufficient essential amino acid (EAA) profiles). For cricket meal, as one of the new high-quality insect protein sources, the crude protein concentration is comparable to that of fish meal. Recent studies have shown that cricket meal can replace part of fish meal, and have achieved good results in Clarias gariepinus, Micropterus salmoides, and other aquatic animals. However, the application of cricket meal as a substitute for fish meal in the diets of yellow catfish has not been reported. In the present study, we investigated the effects on the growth performance, muscle composition, and serum biochemical indexes of yellow catfish by replacing different proportions of fish meal in the diets with cricket meal. The aim was to explore the feasibility of replacing fish meal in the diets of yellow catfish, and to provide a scientific reference for the future development and application of insect protein sources in aquatic feed. The cricket meal used in this study was a brown powder containing dry matter crude protein content of 63.40%, crude fat content of 15.50%, and crude ash content of 7.36%. Healthy juvenile yellow catfish with an average body weight of (2.0±0.13) g were randomly divided into five groups with three replicates and 30 fish per replicate. Five isonitrogenous and isoenergetic experimental diets were formulated by replacing 0%, 15%, 30%, 45%, and 60% of fish meal protein with cricket meal, named T0, T15, T30, T45, and T60 groups, respectively. The experimental fish were reared in an indoor recirculating aquaculture system for 10 weeks. By measuring growth performance, muscle amino acid content, and serum biochemical parameters, the appropriate replacement level of cricket meal in yellow catfish diets was investigated. The results showed that with increasing cricket meal content, the final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) of juvenile yellow catfish increased first and then decreased. The growth performance of FBW, WGR, and SGR in the T30 group was the highest and significantly higher than that of FBW, WGR, and SGR in the T0 group (P < 0.05), whereas the feed conversion rate (FCR) was significantly lower than that of T0 and T15 groups (P<0.05). The hepatosomatic index in the T30 group was higher than that in the T0 and T15 groups (P<0.05), and there was no difference between the T45 and T60 groups. There were no significant differences in the viscerosomatic index, feed intake, FCR, and survival rate among all groups (P>0.05). The EAA contents of the muscle arginine and valine in the T60 group were significantly higher than those in the T0 group (P<0.05). There were no significant differences in the contents of total flavor amino acid in muscle among all groups with different proportions of replaced cricket meal (P>0.05). Compared with the T0 group, the content of glucose (GLU) in the serum of the T30, T45, and T60 groups significantly increased (P<0.05), whereas the content of total cholesterol (TCHO) was significantly decreased (P<0.05). The activities of the serum superoxide dismutase and catalase in the T30 and T60 groups were significantly higher than those of the T0 group (P<0.05). In conclusion, under our experimental conditions, the growth performance and muscle amino acid composition of juvenile yellow catfish were not affected by replacing fish meal with cricket meal, and serum biochemical parameters and TCHO contents were increased. The optimal growth rate was achieved by replacing fish meal with 30% cricket meal. The results of indicate that cricket meal is an excellent substitute for fish meal and provides a theoretical reference for the application of cricket meal as a partial substitute for fish meal in aquatic animal diets.

Abstract:To investigate the effects of replacing fish meal with fermented Antarctic krill meal on growth performance, body color, and serum biochemical indexes of koi carp, 450 koi carp with an average body weight of (4.92±0.22) g were randomly divided into five groups with three replicates per group and 30 fish per replicate. In the control group, we added unfermented defatted Antarctic krill meal, and in the experimental groups, we added defatted Antarctic krill meal fermented by Enterococcus faecalis, Lactobacillus plantarum, Clostridium butyricum, and compound bacteria (1:1:1), which were termed as ES (control group), EF, LP, CB, and MIX, respectively. The addition amount of Antarctic krill meal in each group was 200 g/kg, and the experiment lasted for 70 days. The results showed the following: Compared with those in the control group, the replacement of fish meal with fermented Antarctic krill meal significantly improved final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) (P<0.05); additionally, the FBW, WGR, and SGR of the MIX group were significantly higher than those in other groups (P<0.05). The carotenoid content in the skin and scales of the experimental group was significantly increased (P<0.05); the red (a*) and yellow (b*) value, and relative expression of tyr in the skin and scales were significantly increased (P<0.05); and the a* and b* values and the relative expression of the tyr gene in the MIX group were significantly higher than those in other groups (P<0.05). The contents of serum triglyceride (TG) and total cholesterol (TC) in the experimental group were significantly increased (P<0.05), whereas the ratio of glutamic oxalate transaminase/alanine transaminase (AST/ALT) and the content of malondialdehyde (MDA) were significantly decreased (P<0.05). The liver cells of the control group showed lipid droplets and vacuolar degeneration. No significant difference was observed in each index among the EF, LP, and CB groups (P>0.05). Based on the above results, using complex bacteria to ferment krill meal in the koi carp feed is recommended with a 20% addition level of defatted krill meal, which can effectively improve the absorption and conversion utilization rate of krill meal.

Abstract:The red swamp crayfish (Procambarus clarkii) is the largest freshwater shrimp cultured in China. Protein is a critical factor affecting red swamp crayfish growth performance and feed cost. Too high or too low protein levels in feed negatively affect crustaceans. However, the results of the optimal dietary protein content of red swamp crayfish identified in different studies vary considerably. There are mainly two types of feed for red swamp crayfish on the market: extruded and pellet feed. There have been few reports on the impact of processing technique on red swamp crayfish. Many studies have shown that the growth-promoting effects of extruded and pellet feed on diverse aquatic animals differ considerably. Moreover, there have been few studies on the impact of different protein levels and processing techniques on the growth of aquatic species. This study aimed to investigate the effects of dietary protein levels and processing techniques on the growth performance, digestibility, and antioxidant capacity of red swamp crayfish. The two-factor experiment (4×2) with four different protein levels (28%, 30%, 32%, and 36%) and two processing techniques (expanded feed [EF] and pellet feed [PF]) was conducted for 13 weeks as a feeding trial, aiming to provide theoretical support for optimizing the feed formula and processing technique of red swamp crayfish. The results showed that the protein levels and processing technique significantly influenced the weight gain rate (WGR) and hepatosomatic index of red swamp crayfish (P<0.05). The main effects showed that 28% protein could significantly increase the WGR of red swamp crayfish. The WGR and SGR (specific growth rate) of the EF group were significantly higher than those of the PF group (P<0.05). There was an interaction between the protein level and processing technique and the trypsin and amylase activities in the hepatopancreas of the red swamp crayfish (P<0.05). The activities of trypsin and amylase in the hepatopancreas and intestine were highest, with 28% of dietary protein. Trypsin and amylase activities in the hepatopancreas and trypsin activity in the intestine of red swamp crayfish from the EF group were higher than those from the PF group (P<0.05). The impact of dietary protein levels and processing technique on the malondialdehyde (MDA) content and alkaline phosphatase (AKP) activity in the hepatopancreas of the red swamp crayfish was observed (P<0.05). The results revealed that AKP activities in the hepatopancreas, and AKP and acid phosphatase activities in the serum were significantly lower at 28% dietary protein (P<0.05). The MDA content in the hepatopancreas of the PF group was significantly lower than that in the EF group, respectively (P<0.05). The AKP activity in the hepatopancreas of the PF group was significantly lower than that of the EF group (P<0.05). Logistic, Gompertz, von Bertalanffy, and Brody models could be suitable for fitting the growth curves of body weight and body length of red swamp crayfish with a R2 value of no less than 0.97. The Logistic model was the most suitable for fitting the body weight and length, and the power function could reflect the relationship between the body weight and length. Overall, a dietary protein level of 28% and expanded processing could optimize the growth performance and digestibility of red swamp crayfish, and supplementation with a 28% dietary protein level and pellet processing could improve their antioxidative ability.

Abstract:The transcription factor activating protein 2α (AP2α) is a nuclear transcription factor that specifically binds to DNA and is involved in the regulation of animal embryonic development, cell growth, apoptosis, tumorigenesis, immunity, and other biological processes. In our previous study, the transcription factor AP2α was discovered as a key disease-resistance candidate gene in the yellow drum, Nibea albiflora, in response to a Vibrio harveyi infection through genome-wide association analysis. In the present study, the AP2α gene, which encodes a protein of 424 amino acids, was cloned from a yellow drum. The N-terminal is a trans-activation domain rich in proline and glutamine (P/Q-rich domain), and the middle is a central basic structure (central basic region), which is a highly conserved helix-span-helix motif responsible for DNA binding and protein dimerization. Multiple alignments of amino acid sequences showed that AP2α was highly conserved, with a homology of more than 84.63% among the detected fish, amphibians, birds, and mammals. Quantitative RT-PCR demonstrated that AP2α was widely distributed in the nine tested tissues, with the highest expression in the blood. Moreover, its transcription was significantly activated in the liver, spleen, and head kidney by V. harveyi infection, especially in the liver wherein the transcript level of AP2α reached a peak at 24 h post infection. Subcellular localization by constructing the recombinant eukaryotic expression plasmid pGEFP-AP2α and transfection into HEK293T cells revealed that AP2α was localized in the nucleus. In addition, the soluble GST-AP2α fusion protein was expressed in Escherichia coli BL21. These results demonstrate that AP2α plays an important role in the immune response against V. harveyi in N. albiflora. We provide new insights into the role of AP2α in the innate immunity of teleost fishes and provide a basis for studies on immune mechanisms and disease-resistant breeding in N. albiflora and other marine fish.

Abstract:The skeletal system of fish consists of the axial skeleton (skull, vertebral column, ribs, and intermuscular bones) and the appendicular skeleton, which are essential for behavioral and physiological functions such as locomotion, feeding, predator avoidance, and load-bearing. As for the vertebral column of teleosts, it is composed of many vertebrae connected from the head to the caudal base. The morphological characteristics of the vertebrae (such as the number and structure) vary among different fish species. These characters (especially the vertebrae number) provide an important basis for species identification. For instance, the number of vertebrae in Salmo salar is 57–60 (30 trunk vertebrae, 27–30 caudal vertebrae), while rainbow trout (Oncorhynchus mykiss) has a total of 63 vertebrae (including 33 trunk vertebrae and 30 caudal vertebrae), which can be used for species identification. Fish with a similar number of vertebrae require further skeletal morphological features to distinguish them. For instance, the three-dimensional structure of the same vertebra segment from 32 different teleost species (belonging to 10 different orders) were compared and analyzed using Micro-computed tomography (Micro-CT) scanning technology. The results showed that the lamellar trabeculae and its internal cavity structure on the spine differed between the species, suggesting that these structural characteristics can serve as additional evidence to classify and identify fish species. In addition, calcium (Ca) and phosphorus (P) are the most important mineral elements in a fish skeleton, and their contents vary among different fish. Therefore, there is potential to use the skeletal Ca and P contents in classifying and identifying fish and their life history characteristics. To examine the vertebrae number in O. mykiss, specimens of juvenile O. mykiss of body weight (1.27±0.21) g were cleaned and double-stained to obtain the whole skeletal image. A total of 63 vertebrae were identified, and all were completely ossified at this developmental stage. X-ray scanner technology scanned and photographed the entire skeletal structure of adult O. mykiss. The adult results were similar to the juvenile results of 63 vertebrae with both ends connected with the head or tail, and the ribs were attached to the trunk vertebrae. The ventral sides of the 1–33 trunk vertebrae were arranged in an arc, which was downward and separate. No intermuscular spine was evident. On the dorsal side of the vertebrae, the neural arches surrounding the neural canal were fused with the neural spines. Unlike the ribs, the caudal vertebrae had vascular arches, which formed passages for blood vessels and nerves and were fused with the vascular spines on the ventral side. The calcium and phosphorus content, and microstructure of the vertebrae in O. mykiss at different developmental stages were assessed. Vertebrae samples were collected at four developmental stages (young stage Ⅰ, young stage Ⅱ, adult stage Ⅰ, and adult stage Ⅱ; with an average body weight of 4, 35, 644 and 2 129 g, respectively). The calcium and phosphorus contents in the 1–6th vertebrae were assessed by inductively coupled plasma mass spectrometry (ICP-MS). The 4–6th vertebrae were scanned using Micro-CT technology. The results revealed the calcium and phosphorus contents of the vertebrae initially increased and then decreased during development. The highest levels of calcium and phosphorus in the vertebrae was at young stage Ⅱ, (4 711.121±567.948) and (3 649.488±446.961) μmol/g, respectively. The Ca/P molar mass ratio increased significantly with the growth of O. mykiss (P<0.05).猠⁔獨楥杳湥椠晲楥捳慵湬瑴汳礠⁩摮畤物楣湡杴⁥摤攠癴敨污潴瀠浴敨湥琠⁤慥湧摲⁥瑥栠敯⁦爠敭汩慮瑥楲癡敬⁩牺敡獴畩汯瑮猠⁩据漠畴汨摥†灶牥潲癴楥摢敲⁡浥漠物敮⁣牲敥污楳慥扤氠敷⁩摴慨琠慧⁲景潷牴⁨愠条敮Ɽ†杤牥潶略灬Ɐ⁰慭湥摮⁴琮愠硍潩湣潲浯椭捃⁔椠摳散湡瑮楮晩楮捧愠瑲楥潳湵潴晳†晩楮獤桩⹣੡ted that the bone volume and surface of the vertebrae increased significantly with the growth of O. mykiss. The vertebrae segments became more obvious, and the structure became more complete. The vertebral microstructure indexes in O. mykiss at the different developmental stages suggested the trabecular number (Tb.N) significantly decreased with the growth of O. mykiss (P<0.05). The highest levels occurred at young stage Ⅰ with (19.915±0.758) ind./mm, the lowest levels occurred at adult stage Ⅱ with (1.960±0.043) ind./mm. The trabecular thickness (Tb.Th) and trabecular separation/spacing (Tb.Sp), both significantly increased with O. mykiss growth (P<0.05). Tb.Th and Tb.Sp of the vertebrae in O. mykiss were the lowest, (0.060±0.001) mm and (0.068±0.004) mm, respectively at young stage Ⅰ, and the highest levels, (0.718±0.026) mm and (0.402±0.029) mm, respectively) were at adult stage Ⅱ. In addition, the bone volume fraction (BV/TV), tissue mineral density (TMD), and bone mineral density (BMD) showed a trend of initially decreasing and then increasing. The lowest levels were at adult stage Ⅰ, (62.620±13.223)%, (460.300±102.825) mg/mL and (678.052± 4.417) mg/mL, respectively, and the highest BV/TV and TMD, (86.473±1.029)% and (654.797± 7.031) mg/mL were at adult stage Ⅱ. Conversely, the highest BMD, (820.527±5.003) mg/mL, was at young stage Ⅰ. The evaluation indexes of the bone spatial morphological structures (such as TV, BV, BV/TV, BS, Tb.Th, and Tb.Sp) increased significantly during the growth and development of rainbow trout, while Tb.N decreased significantly. The bone strength evaluation index, BMD initially decreased and then increased. The significant variation in the vertebra microstructure at the different developmental stages might be closely related to its function. These results indicate that the microstructure and element contents of vertebrae in O. mykiss change

Abstract:The Chinese longsnout catfish (Leiocassis longirostris) is a rare and valuable freshwater fish wildly distributed throughout China. Fish growth is one of the most economically important traits in fish farming. Cultured fish with high growth performance can often bring direct economic benefits while meeting human food demand. The hypothalamus is an important regulatory organ in fish metabolic processes and endocrine activities, directly or indirectly regulating fish growth. Although significant research on L. longirostris has been reported, the molecular mechanisms and key genes involved in its growth are still unclear. Therefore, we performed comparative transcriptomics analysis using Illumina high throughput sequencing technology and analyzed transcript profiles of the brains from fast-growth (FG) with average body mass of (534.02±53.68) g, and slow-growth (SG) with average body mass og (108.41±4.96) g L. longirostris individuals. A total of 267 404 674 clean reads were generated, and 518 differentially expressed genes were identified, of which 412 genes were up-regulated and 106 genes were down-regulated in fast-growth fishes. Then, we subjected all these differentially expressed genes to GO term enrichment and KEGG pathway analysis to find the underlying function annotation. Based on Gene Ontology analysis, plenty of differentially expressed genes were enriched in growth, growth factor activity, and hormone-mediated signaling pathway. KEGG enrichment analysis indicated that some differentially expressed genes involved in MAPK signaling pathway, TGF-beta signaling pathway, calcium signaling pathway, and neuroactive ligand-receptor interaction were enriched. With the differentially expressed genes identified from GO and KEGG enrichment analysis, several key genes related to the growth of L. longirostris were screened, such as gnrh, thr, egr1, fgf18, sst, gipr, cart, and crf. The results of this study enriched the gene resources and provided valuable references for further study on the regulation mechanism of growth of L. longirostris.

Abstract:Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment. The incredible demand promotes the rapid development of the crayfish breeding industry. Viral diseases caused by white spot syndrome virus (WSSV) are widely spread in crustaceans, including P. clarkii. WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality. Virus infection can directly induce autophagy mechanisms. Autophagosomes can wrap virus particles and transport them to lysosomes for degradation. As a highly conserved cellular defense mechanism, autophagy plays an important role in the regulation of virus infections. However, many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication. In this study, WSSV in susceptible P. clarkii were explored to determine how autophagy related genes of P. clarkii participate in the regulation of virus infection. To study the role of the autophagy related gene (Atg2) in the innate immunity of P. clarkii, the full-length sequence of the Atg2 gene in P. clarkii (named PcAtg2) was cloned using the total RNA of P. clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique (RACE). The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P. clarkii was 9 966 bp, including a 582 bp 5' non coding region, 2 817 bp 3' non coding region, and 6 567 bp open reading frame. We speculate it encodes 2 189 amino acids. Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family, with 65 serine phosphorylation sites, and 48 glycosylation sites. The amino acid sequence of PcAtg2 in P. clarkii had the highest homology with the Homarus americanus Atg2 gene. The distribution of the PcAtg2 gene in the gill, heart, midgut, hepatopancreas, stomach, muscle, hemocyte, epidermis, testis, ovary, abdominal ganglion, and eyestalk of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that there was no significant difference in the expression of the PcAtg2 gene between male and female individuals. However, there were variations in expression in the different tissues. PcAtg2 was expressed in all tissues of P. clarkii, with the highest expression in the hepatopancreas and the lowest expression in the eyestalk. Under WSSV infection, PcAtg2 was initially up-regulated and then down-regulated in the different tissues, after induced expression. These findings suggest that PcAtg2 is involved in the regulation of autophagy in P. clarkii infected with the WSSV virus, and also plays an important regulatory role in the immune response. RNA interference (RNAi) technology was used to further explore the autophagy related genes PcAtg2 of P. clarkii and their role in WSSV infection. In the WSSV infection experiment with P. clarkii, the copy number of the WSSV virus in the dsPcAtg2 injection group was significantly lower than that in the control group and the dsGFP injection group, indicating that the replication of the WSSV virus was inhibited to some extent during the gene silencing of PcAtg2. The mortality results also showed that silencing PcAtg2 could reduce the mortality of P. clarkii infected with WSSV. In this experiment, after PcAtg2 was silenced, the transmission electron microscope images showed that after 24 and 48 hours of WSSV stress, autophagy vacuoles began to appear in the lysosomes in the hepatopancreas of P. clarkii in the control group, the injected dsPcAtg2 group, and the dsGFP injected group. More autophagosomes appeared and accumulated near the nucleus, indicating that P. clarkii can activate the regulation of cell autophagy under WSSV stress. Among them, more autophagosomes appeared in the hepatopancreas of P. clarkii in the dsPcAtg2 injection group, indicating the PcAtg2 gene promoted the formation of autophagosomes. WSSV virus proliferation can take advantage of autophagy. To avoid using the virus, cells will relatively down regulate the expression of autophagy related genes, and reduce the level of autophagy. In this experiment, by silencing the expression of the PcAtg2 gene, P. clarkii can promote autophagy regulation by up-regulating the expression of other autophagy related genes. In conclusion, the full-length sequences of the autophagy related gene PcAtg2 in P. clarkii were obtained for the first time, allowing us to reveal the effect and mechanism of WSSV infection on autophagy in P. clarkii. The effect of regulating autophagy on WSSV replication was analyzed, and the mechanism of the PcAtg2 gene acting on virus replication by regulating the formation of autophagosome was clarified. The PcAtg2 gene plays an important role in anti-virus immune defense in P. clarkii. We provide a theoretical basis for investigating anti-virus strategies from the perspective of autophagy. Further research on the host defense mechanism regulated by autophagy will provide new antiviral strategies.

Abstract:UVA is an important component of natural light and has certain ecological functions. However, it remains unclear whether UVA affects the nutrient composition of Penaeus vannamei. We built a supplementary light culture system for P. vannamei and used classic nutrient composition analysis techniques to analyze the nutrient, fatty acid, and amino acid composition of the shrimp muscle tissue after different supplementary UVA light durations. Our results provide a theoretical reference for the technological improvement of P. vannamei culture. A total of 450 shrimps [weighing (9.56±0.10) g] were included in a 28-day culture experiment with background lighting of 12L:12D photoperiod with full spectrum LED light [light intensity (1.00±0.02) W/m2]. The experimental design randomly included different UVA [light intensity (1.00±0.02) W/m2] supplementation (0 h, T0 h; 2 h, T2 h; 4 h, T4 h; 8 h, T8 h; 12 h, T12 h). The results revealed no significant variation in the water and crude ash content of the shrimp muscles after different UVA light durations. The crude fat content increased significantly in the T2 h and T4h groups (P<0.05). The crude protein was significantly higher in the T2h group than that in the other groups (P<0.05), except the T4h group. The crude fat was significantly lower in the T8 h and T12 h groups than that in all other groups (P<0.05), but there was no significant difference between the T8 h and T12 h groups. Among saturated fatty acids (SFA) in the shrimp muscle, C16:0 was highest (with 17.19%–27.03%), followed by C18:0 (with 7.82%–10.99%), and both were significantly higher in the T2 h and T4h groups (P<0.05). The dominant monounsaturated fatty acids were C18:1n-9 (with 8.46%– 14.21%), and were significantly higher in the T2h and T4h groups (P<0.05). Linoleic acid and EPA were significantly higher in the T2 h and T4 h groups (P<0.05). The SFA content was 27.85%–40.70%, MUFA was 10.63%–16.31% and PUFA was 16.31%. The total content of n-3 and n-6 polyunsaturated fatty acids was significantly higher in the T2 h and T4 h groups than those in the other groups (P<0.05), and was significantly lower than those in the T12 h group, and significantly low than those in the T8 h group (P<0.05). However, the difference between the T12 h and T8 h groups was not significant (P>0.05). Seventeen common amino acids (excluding tryptophan, which was not detected) were detected in the muscle of P. vannamei. These included seven essential amino acids (EAA), two semi-essential amino acids (HEAA) and eight non-essential amino acids (NEAA). The results showed significant variation between the fraction of the 17 amino acids at different UVA light supplementation durations, with three essential amino acids (methionine, leucine, and lysine), one semi-essential amino acid (arginine), three non-essential amino acids (aspartic acid, glutamic acid, and glycine) and total, essential, semi-essential and non-essential amino acids in the T2 h and T4 h groups. The content of the three essential amino acids (threonine, isoleucine, and phenylalanine) did not differ significantly (P>0.05) with the different UVA light supplementation durations. The amino acid composition in the shrimp muscles showed that among the 17 amino acids at different UVA supplementation durations, the highest levels were glutamic acid with 2.02%, 2.72%, 2.71%, 1.95% and 1.93% in the T0 h, T2 h, T4 h, T8 h, and T12 h groups respectively, followed by glycine, aspartic acid, arginine, leucine, and lysine. The cystine content was the lowest, at 0.02%, 0.03%, 0.04%, 0.03% and 0.04% in the T0 h, T2 h, T4 h, T8 h, and T12 h groups, respectively. The EAA/TAA of shrimp muscle at different UVA supplementation durations ranged from 0.36 to 0.38 and EAA/NEAA from 0.68 to 0.73, with no components varying significantly between the treatments (P>0.05). The evaluation of the nutritional composition of the muscle of P. vannamei under different UVA light supplementation durations identified the muscle composition of shrimp in the T2 h and T4 h groups was high in protein and fat, while other nutritional components did not vary significantly from the other three groups. The EAA, HEAA, NEAA, and TAA contents, as well as the C16:0 and polyunsaturated fatty acids were higher in the T2 h and T4 h groups than those in the other groups. Therefore, shrimp in the T2 h and T4 h groups were more nutritious with a better nutritional status. In terms of the nutritional composition of the muscles of P. vannamei, 2–4 h of UVA supplementation can improve their nutritional quality and increase their nutritional value to a certain extent. In conclusion, the aquaculture light environment for P. vannamei requires further optimization.

Abstract:Chinese mitten crab (Eriocheir sinensis) belongs to Decapoda, Grapsidae and Eriocheir. It is also known as river crab and hairy crab and is widely distributed in the coastal waters of China and has important economic benefits. Salinity is the key environmental factor affecting the mating and spawning of E. sinensis. Presently, reports on the effects of salinity on crabs focus on osmotic pressure regulation, nutritional and energy metabolism, molting, and sexual precocity. However, the effects of salinity on the related hormones in the hemolymph before and after mating and spawning of E. sinensis have not been reported. Relevant steroid hormones include progesterone, 17α-dihydroxyprogesterone, 20β-dihydroxyprogesterone, and 17α-20β-dihydroxyprogesterone (DHP) and most effectively induce maturation of salmon and trout eggs, which has been confirmed in a variety of fish. Early studies have shown that the ovulation activity of fish is closely related to the level of gonadotropin (GTH) in the blood. The gonadotropin releasing hormone receptor (GnRHR) in E. sinensis indicate that the ovarian maturation and reproductive regulation in crustaceans are regulated by some gonadotropins, such as methylfarnesol and ecdysone. PGE, PGF, and prostacyclin (PGI2) can promote ovulation in a variety of fish and crustaceans. Like vertebrates, steroids are important hormones that affect the ovarian development of shrimp and crab, such as estradiol (E2), testosterone (T), and progesterone. Steroids can impact gonadal development and play a very important role in ovarian development and vitellogenesis. However, these hormones are restricted by a variety of environmental factors, such as temperature, light, salinity, and so on. In order to investigate the effect of salinity on hemolymph related hormones during mating and spawning of E. sinensis, the spawning salinity was set at 0, 2, 4, 6 and mating salinity set at 3, 6, 9, 12, 15, 18, 21. Five samples were collected from specimens in each salinity to analyze the contents of DHP, GTH, PG, E2, and testosterone in the hemolymph of female E. sinensis before and after mating and spawning under different mating and spawning salinities. The components were detected by ELISA at the wavelength of 450 nm using the kit double antibody sandwich method. Results revealed: (1) in fresh water, female crabs displayed no mating behavior. When the salinity was lower than 6, female crabs only mated without spawning. (2) There were no significant changes in PG, E2, or testosterone in the hemolymph of female crabs after mating in low salinity (2–6), and there was no significant difference between crabs at different salinities after mating (P>0.05). (3) The contents of DHP, PG, E2, and testosterone in the hemolymph of female crabs initially decreased and then increased with salinity before spawning. After spawning, DHP, PG, and E2 in hemolymph initially increased and then decreased with increased salinity. When the salinity was 18, DHP, PG, E2, and testosterone in the hemolymph of the female crab after spawning decreased to the lowest level, and there was a significant difference between the before and after spawning results (P<0.05). (4) At 6 salinity, the contents of DHP, PG, E2, and testosterone in the hemolymph of female crabs before spawning were the highest and the contents of DHP and PG decreased after spawning, while the contents of E2 and testosterone increased. However, there was only significant variation in the DHP from before spawning to after spawning (P<0.05), and there was no significant difference in the other indexes (P>0.05). The content of testosterone in the hemolymph initially decreased and then increased before and after spawning. This comprehensive study showed that salinity effects the five hormones in the hemolymph of female E. sinensis during the reproductive and breeding stage. Among them, the five hormones do not change significantly during the mating process of E. sinensis. DHP, GTH, and PG are involved in the oviposition of E. sinensis. The analyses of the changes in the related hormones in the hemolymph of female E. sinensis before and after mating and spawning at different salinities have identified changes in the neutral hormones. These results provide details on the reproductive regulation mechanism of E. sinensis and provided basic reference data for researching the reproductive biology of E. sinensis.

Abstract:Mud crab reovirus (MCRV) is one of the most fatal pathogens of the mud crab Scylla paramamonsain. The outbreak and epidemic of MCRV has seriously affected the healthy development of the mud crab aquaculture industry. To limit MCRV transmission from breeding crabs to larva, we attempt to establish a more sensitive and practical diagnostic method for screening MCRV-free crabs. The primers of the present diagnostic methods for MCRV are based on the VP1 gene (MCRV RNA polymerase gene), and the low expression level of this gene limits the sensitivity of the diagnostic method. Therefore, it is necessary to select the target gene with the highest expression level for the detection primer to improve the sensitivity of the diagnostic method. In addition, the current diagnostic methods require gill samples for virus detection, which requires killing the mud crabs before sampling. This sampling method is obviously not suitable for screening MCRV-free breeding crabs. Therefore, it is necessary to develop a less invasive sampling method for breeding crabs. In this study, a SYBR Green fluorescent quantitative diagnostic method was developed to screen MCRV-free breeding crabs. To improve the sensitivity of the detection, we initially analyzed the relative load of MCRV in the main tissues of infected mud crabs. The viral load in the hemolymph was the highest of all the tissues. The expression levels of 13 putative genes of MCRV were detected in the hemolymph. The relative expression level of the VP11 gene was the highest. Finally, specific primers were designed based on the conserved region of the VP11 gene sequence to establish a SYBR Green qRT-PCR (quantitative reverse-transcription PCR) detection method to accurately detect 50 copies/µL of viral nucleic acid in a sample. Considering the advantages of tissue and target gene selection, the sensitivity of this method should be significantly higher than that of preexisting detection methods. This diagnostic method is very specific for MCRV and no specific amplification was observed using nucleic acid samples containing 5 different kinds of common crustacean pathogens (MCDV, WSSV, DIV1, EHP, and Vibrio parahemolyticus). Compared to other methods of extracting RNA by killing and grinding the gill tissues of crabs, we can select MCRV-free crabs by sampling very small amounts of hemolymph (as low as 20 µL). All of the healthy crabs screened by this method were able to hold eggs that hatched normally. To test the effectiveness of this method, 22 breeding crabs and 20 commercial crabs were screened for MCRV. The positive rates were 54.55% and 85.00%, respectively. In addition, we analyzed the proliferation of MCRV in the mud crabs, and found that MCRV proliferates exponentially in the early stage, then enters a plateau phase, and no crabs died during the infection period of seven days. In conclusion, this study established a highly-sensitive and practical detection method for MCRV in breeding crabs, which can meet the requirements for MCRV-free breeding crab screening with low damage to the breeders. We also investigated the pathogenic infection mechanisms.

Abstract:The East Asian common octopus (Octopus sinensis), is mainly distributed along the southeast coast of China. It has become an important aquaculture species in the coastal areas of the Fujian and Guangdong province due to its excellent culture features such as high conversion rate, fast growth rate, large size, and high market price. The main culturing methods of O. sinensis are industrialized, indoor, and cage culturing. The density is high during cage culturing. If the feed is insufficient or the feeding interval too long, there are attacks and cannibalism among individuals, which adversely impacts the culturing income. Therefore we urgently need to undertake digestive physiology research to explore the digestive functions of O. sinensis. This research will provide valuable information to identify an appropriate feeding frequency and reduce the economic loss caused by cannibalism. At present, there is no relevant research on the digestive physiology of O. sinensis. In this study, the histological structure of each digestive organ of O. sinensis was observed to explore the relationship between the tissue structure and digestive function. We investigated the trypsin, amylase, and lipase activities in the anterior salivary gland, posterior salivary gland, crop, digestive gland, stomach, cecum, and intestine before and after feeding. The changes in plasma glucose, plasma triglyceride, plasma total cholesterol, plasma total protein and muscle glycogen before and after feeding were analyzed to determine the digestion and absorption duration in O. sinensis. O. sinensis is an aggressive carnivore with a highly developed digestive system. The histological results showed that the crop, stomach, cecum, and intestine of O. sinensis is composed of three layers, the mucosal, submucosal, and muscular, and the inner wall contained many folds. Both the anterior and posterior salivary glands were compound tubular glands, which were composed of oval and circular gland tubes with many secretory ducts. The crop widens and expands from the esophagus into a large lumen, and the cuticle of the mucosal layer was thin (with a thickness of approximately 2.57–5.44 μm). The stomach was spherical, the gastric cavity was biased to one side, and the inner wall contained many folds. The muscular layer of the gastric wall was the most developed layer with the cuticle approximately 99.97–383.82 μm. The cecum was double helix and the inner wall contained rich long folds and secondary folds. The submucosa at the free end of some of the long lateral folds contained some mucous glands, and the mucosal layer without any cuticle. The digestive gland was the largest proportion of the digestive system and was composed of hepatic lobules. The boundary of the hepatic lobules was difficult to distinguish. The diameter of the intestine was approximately 2.50 mm, the tube wall was thin and the mucosal layer had no cuticle. The intestinal mucosa forms wavy longitudinal folds in the mucosa, including a pair of longer folds, which occupied almost the entire intestine. The analysis of the digestive enzyme activities and nutrient metabolism showed that trypsin activity was significantly higher than that of lipase and amylase. The trypsin activity in the digestive gland reached (148.74±21.25) U/mg, and the amylase activity in the digestive gland and stomach reached (3.68±0.59) U/mg and (2.48±0.64) U/mg, respectively. The lipase activities did not vary between the different tissues. After feeding, the plasma total protein concentration reached the lowest levels of (161.50±67.51) mg/mL at 120 min, and then increased. The plasma glucose and muscle glycogen concentrations of O. sinensis reached maximums of (1.54±0.44) mmol/L and (4.75± 0.13) mg/g at 200 min, respectively. Feeding had little effect on the plasma lipid concentration in O. sinensis, the plasma cholesterol concentration did not vary significantly while feeding (P<0.05), and there was limited variation in the plasma triglyceride concentration. The digestion process was divided into two stages. The first stage occurs in 0~60 min as food entered the crop and stomach, and the digestive gland secreted large doses of trypsin. Initially, the trypsin in the stomach mixed with the food, resulting in decreased trypsin activity. At this stage, only small amounts of nutrients are absorbed and the body needs to consume nutrients to supply energy for the digestion process. Therefore, a decrease in plasma glucose, plasma protein, and muscle glycogen content occurred. The second stage occurs in 120~300 min and involves high trypsin activity. In the progress of extracellular digestion and intracellular digestion, the trypsin activity of various digestive organs decreased gradually, and nutrients increased gradually. At 400 min, the trypsin activity, plasma glucose, plasma protein, and muscle glycogen returned to the pre-feeding levels, indicating the end of the digestion and absorption process. The whole digestion and absorption process of O. sinensis lasted approximately 400 min. Therefore, the appropriate feeding interval for O. sinensis culture is 6~7 h.

Abstract:Rapana venosa is primarily distributed in the Yellow Sea and Bohai Sea of China, Japan, Korea, and Russia. In the natural sea area, the adult R. Venosa mostly inhabits the sand-mud bottom or the rocky bottom of the low intertidal zone up to 20 meters deep, and the young R. Venosa mostly inhabits the rocks near the coastal line. Its habitats are generally occupied by many other bivalves, such as Crassostrea gigas, Mactra chinensis, and Ruditapes philippinarum. The R. venosa is a large carnivorous Mollusca that mainly feeds on bivalves and other animal carcasses. R. venosa is often classified as an enemy of bivalve farming, but they can also be used to control fouling organisms, and it has potential for application in aquaculture and ocean engineering. In recent years, there have been some reports on the feeding selectivity of R. venosa, the effects of feed, temperature, and individual specifications on feeding, the effects of feed types, specifications, feeding amount, and breeding density on the survival and growth, the feeding preferences to different bivalves and the feeding cycle before and after reproduction. These studies mainly explored the effects of temperature, density, feed types, and specifications on its growth and development. The main purpose of these studies was to select suitable feed types and specifications for the temporary culture and breeding of R. venosa, to improve its growth and development speed, and increase economic benefits under artificial breeding conditions. However, the feeding selection and behavior process of R. venosa under natural conditions are not clear, and the feeding selection of R. venosa to C. gigas, Mytilus edulis and other fouling organisms was not clear under the environmental conditions of multiple bait bivalve habitats. Therefore, in this study, four kinds of bivalves (fixed type, attached type and buried type) were used as bait bivalves to understand the feeding selection. C. gigas and M. edulis are common fouling organisms in the habitat of R. venosa, while M. veneriformis and R. philippinarum are widely distributed in the habitat of R. venosa. The study attempts to simulate the habitat of R. venosa and different types of bait bivalves in the natural environment, to study its preference for bait bivalves species and feeding specifications, to compare and analyze the differences in feeding rates of different specifications of R. venosa, and further study its feeding rhythm and feeding process and to provide data reference for the feasibility of using R. venosa to control C. gigas, M. edulis and other bivalves fouling organisms, and improve the feeding habits of R. venosa. To achieve these objectives, three specifications of M. veneriformis, R. philippinarum, C. gigas, and M. edulis live baits were placed in the aquarium by simulating the natural environment. The feeding number, feeding weight, feeding specifications, feeding time, and feeding behavior process of three specifications of R. venosa to different baits were recorded. The experimental results showed that R. venosa fed on all four bivalves. The number and weight of R. venosa that fed on M. veneriformis with different specifications were significantly higher than those that fed on other bivalves (P<0.05), and the feeding index was more than 50%, indicating its appetite. R. venosa fed normally on C. gigas and R. philippinarum, and only a small population of R. venosa fed on M. edulis with large specifications. In terms of the selection of feeding specifications, three specifications of R. venosa preferred large M. veneriformis and small C. gigas (P<0.05). The feeding rate of the large specification R. venosa was 7.15%, which was significantly lower than that of the other two specifications (small specification, 10.98%; medium specification, 9.64%). Under the experimental conditions, the feeding cycle of R. venosa was apparent, feeding activities were carried out every three days, and the feeding time was 2000–2400 at night. The feeding process can be divided into four stages: Unfed stage, search stage, feeding stage, and feeding end. During the feeding process, the R. venosa actively searched for bivalves, removed them from the sand and wrapped them with their proleg, secreted mucus, and smacked them from the shell gap. After sucking the mollusks decomposed by digestive juice, the R. venosa dived into the sand or attached to the aquarium wall. The results showed that under the experimental conditions, R. venosa had a feeding preference for the species and specifications of bait bivalves. R. venosa preferred to eat M. veneriformis and rarely consumed M. edulis. Moreover, its feeding behavior was nocturnal and exhibited periodicity, feeding activity happened every three days in the first half of the night.

Abstract:The Pacific abalone (Haliotis discus hannai) is naturally distributed in the Bohai Sea and Yellow Sea. In China, the Pacific abalone is an important living marine resource. Over the past 40 years, the abalone industry has gradually developed from wild harvesting to aquaculture. Currently, the main cultivation method is long-line culture, especially north-south relay aquaculture. The north-south relay involves transporting abalone cultivated in the East China Sea to the Yellow Sea and Bohai Sea over summer, to avoid extreme temperature stress. Due to the consistent favorable temperatures, this method achieves high survival with a shortened cultivation cycle. The rapid development of this efficient cultivation model has supported a substantial increase in domestic abalone production, exceeding 200 000 tons in 2020. However, the north-south relay aquaculture has several deficiencies, such as a large influx of abalone being supplied to the market over a very limited period with a homogenized flavor. This has led to a sharp decline in price. Abalone grow slowly in the bottom-sowing model in northern waters, however, the quality exceeds that of north-south relay cultured abalone. The optimum growing temperature of Pacific abalone is 10~22 ℃. Bottom-sowing cultivation in the northern waters has a lower seawater temperature, occasionally below 0 ℃. In addition, in long-term north-south relay cultivation, abalone are always in a suitable water temperature environment, reducing the low temperature tolerance of abalone. The increasing investment in recent years in marine ecological protection (such as marine pastures, habitat restoration and abalone habitat creation) and the technological breakthroughs in the cultivation of low-temperature resistant seedlings has enabled the optimization of bottom-sowing culture, reducing many issues, such as high mortality while overwintering, which has been partially solved. However, the impact of both cultivation methods on the nutrient contents and the physiological index of abalone is rarely reported. In this study, the north-south relay and the northern bottom-sowing abalone cultures were investigated. The total sugar, protein, total organic matter, and amino acid content characterized the nutritional value of individuals from both culture methods. The oxygen consumption rate and heart rate identified their low temperature tolerance. We explore the differences in body composition and physiological mechanisms in response to low temperature stress using specimens from both farming methods. The results showed that the total sugar content of the bottom-sowing culture individuals was (3.20±0.00)%, the total organic matter was (27.60±3.70)%, and the essential amino acid content was (4.19±0.09)%, which were significantly higher than those in the individuals from the north-south relay culture (P<0.05). At 24 ℃, the oxygen consumption rates of the bottom-sown abalone and relay cultured individuals were (0.077±0.024) mg/(g·h) and (0.082±0.012) mg/(g·h), respectively. The oxygen consumption rates of abalone in low temperature stress did not vary significantly, with (0.018±0.009) mg/(g·h) (bottom-sown abalone) and (0.017±0.006) mg/(g·h) (relay cultured abalone) (P>0.05). At 24 ℃, the heart rates of bottom-sown and relay-cultured abalone did not vary significantly, with (45.05±6.79) and (46.95±5.01) BPM, respectively (P>0.05). In low temperature, the heart rate of the bottom-sown abalone was (12.82±1.72) BPM, and the heart rate of the relay-cultured abalone was (18.11±2.79) BPM, statistically differing significantly (P<0.05). The results indicate variation in the abalone responses to external low temperature stress between individuals from the different farming models. The low heart rate level in low temperature conditions indicates a low metabolic level, which can reduce energy consumption, improving survival in the low temperature stress of a northern winter. Studies have revealed different farming models can significantly affect the nutritional value of abalone and the physiological responses to low temperature stress. Abalone cultured by bottom-sowing have higher nutritional value and a low temperature tolerance. In addition, abalone heart rate is a highly sensitive indicator for studying physiological responses to low temperature stress in abalone and other shellfish.

Abstract:At present, the research on the physiological effects of transportation activities on aquatic organisms mainly focuses on fish, crustaceans and echinoderms. There are lacking details for transportation effect on Pteria penguin. In order to explore the effects of transportation modes on the survival, growth, digestion and antioxidant properties of P. penguin juveniles, this study was carried out under the conditions of transportation with water and without water, respectively. The experiments were carried out under the conditions of two modes of transportation with water and without water for 8 hour, respectively. The water temperature was controlled at (20.0±2.0) ℃ for transportation with water, while the temperature in the waterless styrofoam box was controlled at (15.0±3.0) ℃. The culture was temporarily maintained for 14 days after transportation. The breeding conditions and management methods were basically the same before and after transportation. The water temperature was (27.5±1.0) ℃, and the salinity was (31.5±0.5). Every day change 2/3 of the water and feed the mixed algae liquid of Isochrysis zhanjiangensis, Chaetoceros muelleri and Platymonas subcordiformis. The survival rates and growth parameters were estimated after 8 h transportation, 7 days and 14 days temporarily maintained, respectively. The P. penguin juveniles were sampled before transportation and used as control group, then juveniles were divided into two experimental groups for transportation with and without water respectively. The juveniles from different experimental groups were randomly sampled after transportation. Then, the juveniles were sampled on the 7th day and 14th day of the recovery period. The activity of amylase (AMS), superoxide dismutase (SOD), acid phosphatase (ACP), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and the content of cortisol were used as biochemical indicators. The frozen soft tissues were dissected on the ice surface and homogenized on ice in 0.2 mol/L (w/v) ice-cold physiological saline, and the homogenates were centrifuged at 2 500 r/min for 10 min. Take the supernatant for enzyme activity determination, and repeat the determination 3 times for each sample. The results showed that the survival rates with water transport and waterless transport were (98.00±0.57)% and (97.00±1.00)%, respectively. On the 14th day of the recovery period, the survival rates with water transport and waterless transport reached (97.00±1.00)% and (82.00±0.71)%, respectively. Furthermore, after 14 days recovery, the shell length, shell height and body weight of P. penguin in water transport were significantly higher than those in waterless transport (P<0.05), while the content of cortisol level was (1 999.50±10.18) µg/L in the P. penguin transported with water, which was significantly higher than those transported without water [(1 668.46±20.36) µg/L]. The amylase activity after both transports increased, and on the 14th day of the recovery period, the amylase activity after transport with water increased to (1.56±0.08) U/mg prot, which was significantly higher than (1.06±0.04) U/mg prot with transport without water (P<0.05). The activities of GOT and GPT were increased in water transportation, while decreasing in waterless transportation. During the recovery stage, the ACP activity was decreased to (79.56±1.04) U/mg prot at 14th day in water transportation group, while increased to (168.24±3.46) U/mg prot in waterless transportation group. Furthermore, the GOT and GPT activities were trends to increase both in water and waterless transportation, while SOD activity was trend to decrease. The research results show that under certain conditions, water transport and waterless transport can significantly affect the growth, digestion and antioxidant properties of juveniles. Under these two transport modes, the water transport effect and the later physiological recovery are relatively better, with higher survival rate and better growth. After transportation, the body of the P. penguin needs a certain period of time to recover to a normal state, and then can be more adaptive to other adverse environmental factors.

Abstract:Undaria pinnatifida is a brown alga with high economic value. Its annual production is second only to Saccharina japonica among economically important brown algae in China. Due to climate change and increased cultivation density, the occurrence of diseases in cultivated seaweeds has become more frequent and serious in recent years. Most diseases are directly or indirectly related to the interactions between the seaweed host and the epiphytic microorganisms such as bacteria. There is a close relationship between algae and epiphytic microorganisms. When the phycosphere niche maintains a dynamic balance, the two rely positively on each other growing and developing together. When the balance is disturbed, the structure of the epiphytic microbial community may change, possibly resulting in algal diseases. The absence of certain microbes may also lead to the failure of key biological processes such as the morphogenesis of the host algae. Therefore, understanding the composition of the epiphytic microbial community is of great significance for the study of the interaction between U. pinnatifida and epiphytic microorganisms and the better control of U. pinnatifida diseases. In addition, stock resources of U. pinnatifida are usually conserved in the form of gametophytes, which can persist for a long time under controlled conditions. Hence, understanding the composition of the epiphytic microbial community will also facilitate the development of efficient conservation methods and help remove microbial contaminations when axenic cultures need to be established. The life history of U. pinnatifida consists of the alternation between heteromorphic macroscopic sporophytes and microscopic gametophytes. The stark morphological and physiological differences between these two phases suggest that the composition of epiphytic microbial communities between them is likely different. However, studies on the composition of epiphytic microbial communities, especially comparison studies between sporophytes and gametophytes in U. pinnatifida are limited. The advent of high-throughput sequencing provides robust and efficient tools for studying the composition and relative abundance of the microbial community associated with the algae. In the present study, sporophytes and gametophytes (each with three biological replicates) of U. pinnatifida derived from the cultivated population in Dalian China were selected as the study objects. After DNA extraction and PCR amplification of the v3–v4 region of 16S rRNA gene and the v4 region of 18S rRNA gene, Illumina HiSeq 2500 high-throughput sequencing platform was used to sequence these specific regions. We identified and classified the composition of epiphytic microbial communities of the gametophytes and sporophytes of U. pinnatifida based on the sequencing results. A total of 446 932 effective reads were obtained through 16S rRNA gene sequencing. The raw reads have been submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/) with the accession number PRJNA823903. The bacterial community composition of gametophyte and sporophyte was revealed to be significantly different, and the diversity of the bacterial community in gametophyte samples was higher than that in sporophyte samples. In gametophytes, Proteobacteria (66.67%) was the most dominant phylum, followed by Bacteroidetes (13.48%) and Cyanobacteria (11.13%). At the class level, Alphaproteobacteria (34.58%) was the most abundant, followed by Gammaproteobacteria (31.01%), Bacteroidia (13.16%), and Oxyphotobacteria (11.13%). Cyanobacteria (95.67%) was predominantly detected in sporophytes, followed by Actinobacteria (1.65%) and Firmicutes (1.48%). The distribution of Oxyphotobacteria, Alphaproteobacteria, Bacteroidia, Gammaproteobacteria, Negativicutes, OM190, Acidimicrobiia, Erysipelotrichia, Planctomycetacia, and Verrucomicrobiae were found to be different between gametophyte and sporophyte samples, among which OM190, Acidimicrobiia and Planctomycetacia were unique to gametophytes. The genus-level bacterial groups detected in gametophyte samples were Lewinella (10.06%), Leucothrix (5.99%), Sulfitobacter (4.06%), Bifidobacterium (0.02%), while Bifidobacterium accounted for 1.41% of the bacterial genus of sporophyte samples. There were 57.37% and 95.68% uncultured bacterium in gametophytes and sporophytes, respectively. We obtained a total of 473 770 effective reads through 18S rRNA gene sequencing. A major share (97.22%) of microeukaryotes in gametophytes were unclassified, while in sporophytes, the number was 94.95%. Streptophyta, Intramacronucleata, Basidiomycota, Apicomplexa, Arthropoda, Bacillariophyta, Chordata, Gastrotricha, Ascomycota, and Mucoromycota were detected both in the gametophytes and sporophytes. Among them, Basidiomycota, Ascomycota, and Mucoromycota belong to fungi. The community abundance of sporophyte samples was higher than that of gametophytes. In gametophyte samples, except for Phaeophyceae (88.77%) to which U. pinnatifida belongs, Copepoda of Arthropoda, Mediophyceae of Bacillariophyta, Mammalia of Chordata, Prostomatea of Intramacronucleata, and Liliopsida were dominant, with a proportion of 0.62%, 0.50%, 0.25%, 0.23%, and 0.23%, respectively. In addition to Phaeophyceae (94.49%), Conoidasida of Protozoa, Agaricomycetes of Basidiomycota, Spirotrichea of Intramacronucleata and Mammalia of Chordata were predominant in sporophytes, accounting for 0.91%, 0.83%, 0.51% and 0.24% of OTUs, respectively. Chytridiomycetes, Nassophorea, Colpodea, Tremellomycetes, Sordariomycetes, Conoidasida, Agaricomycetes, Arachnida, and Chromadorea were only detected in the sporophytes, and there was a significant difference in Spirotrichea abundance between gametophytes and sporophytes. In conclusion, the composition of epiphytic microbial communities and the relative abundance of different bacteria and microeukaryotes in the sporophytes and gametophytes of U. pinnatifida were determined through high-throughput sequencing of the amplicons of the v3–v4 region of 16S rRNA gene and the v4 region of 18S rRNA gene. Remarkable differences were revealed between the two life stages, indicating that their growth and development are associated with different microbial communities. The results of this study provide valuable information for sustainable cultivation and stock culture conservation of this important kelp species.

Abstract:Shellfish are filter feeders that can accumulate toxic algae and their related toxins, increasing risk when consumed. Shellfish toxins can directly affect the physiological activities of marine organisms and threaten the stability of marine ecosystems. Ultimately, these toxins pass through the food chain and can endanger human health and economic security. Globally, shellfish poisoning incidents have occurred in many countries. The Bohai Sea is a semi-enclosed inland sea, where severe eutrophication of the seawater has occurred in recent years, leading to harmful algal blooms. To date, no simultaneous surveillance of diarrhetic shellfish poisonings (DSP) and paralytic shellfish poisonings (PSP) have been reported in the Tangshan shellfish culture area. To better understand shellfish toxin pollution in the shellfish culture areas of Tangshan and the dietary and health risks to residents, Mactra veneriformis, Ruditapes philippinarum, Rapana venosa, Crassostrea gigas, Cyclina sinensis, Meretrix meretrix, Mercenaria mercenaria, and Azumapecten farreri were collected for toxin monitoring from the Tangshan shellfish culture areas in Bohai Sea from October 2019 to September 2020. A total of 34 samples were collected for each shellfish species. Each sample weighed approximately 3 kg. All samples were transported to the laboratory on ice. In the laboratory, samples were flushed with tap water to remove sand and silt and shucked to collect the soft tissue. The tissue was thoroughly homogenized with a household blender, and approximately 50 g of tissue from each sample was stored at –20 ℃ until required for analysis. Five DSP including okadaic acid (OA), dinophysistoxin 1 (DTX1), dinophysistoxin 2 (DTX2), yessotoxin (YTX), and azaspiracid 1 (AZA1), and 14 PSP including saxitoxin (STX), neosaxitoxin (NEO), gonyautoxin 1/4 (GTX1/4), gonyautoxin 2/3 (GTX2/3), decarbamoylsaxitoxin (dcSTX), decarbamoylneosaxitoxin (dcNEO), decarbamoylgonyautoxin 2/3 (dcGTX2/3), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and N-sulfocarbamoyl toxin 1/2 (C1/2) were tested using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The detection limit of the DSP method was 5 μg/kg, and the detection limit of the PSP method was 10–20 μg/kg. The DSP toxins were not detected in any of the samples. Several PSP toxins were detected, including saxitoxin (STX), gonyautoxin 1 (GTX1), gonyautoxin 2 (GTX2), and decarbamoylgonyautoxin 3 (dcGTX3). The GTX1 levels were the highest overall PSP toxin at 537.95 μg/kg in April. The results revealed positive rates of PSP for R. philippinarum, C. gigas, M. meretrix, and M. mercenaria, which were 11.76%, 47.06%, 5.90%, and 8.82%, respectively. Of the toxins tested, none were detected in the remaining samples. The highest PSP toxin levels in the positive samples from R. philippinarum, C. gigas, M. meretrix, and M. mercenaria were 414.26, 532.57, 452.77 and 195.46 μg STXeq/kg, respectively. We ranked the species in order of highest to lowest PSP toxin levels as: C. gigas > M. meretrix > R. philippinarum > M. mercenaria. In general, the toxin content of the shellfish in this area was lower than the EU limit of 800 μg STXeq/kg. The composition of shellfish toxins is related to many factors, including the sampling location and collection time. The toxin accumulation capacity by shellfish is also affected by many factors, including water pollution, salinity, ligh整渠獩畮牴敥瑳桩整⁹搬攠癡敮汤漠灭浯敳湴琠⁩潭晰瑲桴敡獴桬敹氬氠晴楨獥栠⁳慰煥畣慩捥畳氠瑡畮牤攠⁤楥湮摳畩獴瑹爠祯⁦愠湴摨⁥琠潴獸畩灣瀠潡牬瑧⁡捥漠湩獮甠浴敨牥†桳敵慲汲瑯桵⹮੤ing waters. The ecological risk assessment methods used in this study were the risk quotient method (RQ) and the point assessment method. The RQ method is primarily used for semi-quantitative risk assessments to determine whether the pollutant concentrations have harmful effects. The point assessment model is a dietary exposure assessment tool. We applied risk quotient and point assessment methods to determine risk. There was no safety risk in the consumption of shellfish harvested from the Tangshan coastal study area during the study period. According to the point assessment method, at this specific time it was safe to consume the shellfish as the toxin levels were low. This analysis indicated that the safe single intake quantity of shellfish during months with high levels of shellfish-enriched toxins was reduced. As the toxin levels accumulating in different shellfish tissues can vary greatly, each sampled tissue was analyzed separately. The results indicate when there is a high accumulation of shellfish toxins present, consumers should restrict their consumption to a single serving rather than regularly consuming shellfish as part of their daily diet. The safety risk assessment results indicate that the seven shellfish species posed no food safety risk during the study period. This study provides a scientific basis for improving shellfish management practices to ensure shellfish are safe for consumption. This study analyzed the effects of toxin residues in shellfish species; different seasons and different locations vary in toxin content and components. We recommended consumers regulate their consumption to avoid potential poisoning events. This study provides social, economic, and ecological benefits in promoting green and healthy aquaculture of shellfish products, by ensuring the safety of shellfish products for consumer health. However, continuous long-term monitoring of both phytoplankton and biotoxins are recommended to