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Abstract:Bohai Bay is an important spawning and nursery habitat for a variety of economically important fish inhabiting the Yellow and Bohai seas. Based on a meta-analysis of historical data spanning 40 years, combined with a field survey of current fish habitat conditions conducted in Bohai Bay, a long-term dataset containing early life resource surveys (including 53 voyages, from 1982 to 2021) of marine Osteichthyes was built. Based on statistical analyses, long-term trends in ichthyoplankton assemblage structure, biodiversity, and synchrony were interpreted. The results indicate that ichthyoplankton assemblage structure and the center of gravity for spawning and nursery habitats in Bohai Bay are currently experiencing continuous replacement. Seasonal variations in ichthyoplankton assemblage structure, abundance index, predominant taxa, and species diversity levels are evident. Spawning and nursery habitats are concentrated in the central and inner part of Bohai Bay west of 118º30'E. Compared with survey results from the 1980s, the taxonomic composition and abundance of the ichthyoplankton assemblage structure have changed considerably. The function of Bohai Bay as a spawning and nursery habitat for traditionally economically important fish has declined significantly. The abundance index and taxon number of ichthyoplankton in Bohai Bay fell to a historic low in the early 2010s, then rebounded significantly. The taxa number of ichthyoplankton decreased from 39 in the 1980s to 31 in the early 1990s, 34 in the late 1990s, and 22 in the 2000s to pre-2010s, then further decreased to 21 in the early 2010s. From the middle 2010s, it recovered to a certain degree, increasing to 40 species just prior to 2020. The current number of pelagic egg taxa is 22, 80% of the number in the 1980s, and 88% of the abundance in the 1980s. The current (2020—2021) number of larval fish taxa is 26, almost identical to that in the 1980s, with an abundance 1.33 times greater than that in the 1980s. Interannual and interdecadal variations in fish egg and larvae species diversity fluctuated drastically. Interannual taxon substitution was noticeable. However, the substitution rate has increased significantly in recent years. Spawning, habitat, and temperature adaptation studies of breeding stock indicate that the number of taxa first decreased, then increased. The annual proportion of the number of taxa to pelagic eggs decreased, while the proportion of the number of taxa to adhesive eggs and eggs with egg membrane filaments increased. The annual proportion of continental shelf pelagic-neritic fish taxa increased, while the continental shelf demersal and benthopelagic taxa decreased. The ecological density of numbers in the early life history (EDN-ELH) of Konosirus punctatus, Sardinella zunasi, Larimichthys polyactis, Trichiurus japonicus, Lateolabrax maculatus, and Cynoglossus semilaevis decreased significantly, whereas the EDN-ELH of Engraulis japonicus, Scomberomorus niphonius, Thryssa kammalensis, and Sillago japonica increased. Ichthyoplankton abundance in Bohai Bay mainly depends on environmental conditions in the spawning habitat, fishing intensity, and the degree of damage to the early life stages of fish resources. The temporal variation and succession of the ichthyoplankton assemblage structure in Bohai Bay were the specific manifestations of the multidimensional niche disturbance and structural performance deterioration of the fishery resources under the dual disturbance of global warming and overfishing.

Abstract:The ichthyoplankton stage is the stage that is most vulnerable to changes in the marine environment in the development cycle of marine fish. Subtle changes in the marine environment have a strong impact on fish survival, development, and growth. The abundance of fish eggs directly affects the early recruitment of fish resources and determines the vitality of generations. As one of the important spawning grounds of Engraulis japonicus, it is of great significance to understand the distribution patterns and relationship between environmental factors in offshore waters of the Liaoning Province in the North Yellow Sea. Based on the spawning grounds survey carried out in offshore waters of the North Yellow Sea from April to December, 2021, first, Garrison's distribution center of gravity was used to analyze the core spawning ground of E. japonicus and its migration route. Second, a generalized additive model based on Tweedie distribution (Tweedie-GAM) was applied to convey the main drivers of the distribution patterns of the spawning grounds. The relationship between E. japonicus egg density and six natural environment factors of seawater surface temperature (SST), seawater surface salinity (SSS), seawater surface chlorophyll a concentration, zooplankton abundance, phytoplankton abundance, and depth, as well as the factors of time (month) and space (longitude and latitude) were interpreted. Finally, the cross-validation method was used to validate the model and predict the potential spawning grounds. The results showed that the spawning period of E. japonicus was long, lasting from April to November, and the main spawning period was from May to August, with peak spawning from late May to early June, in offshore waters of the Liaoning Province in the North Yellow Sea. During the spawning season, the size and location of the E. japonicus spawning grounds showed obvious spatiotemporal variation. There was a significant nonlinear correlation between spatiotemporal factors and the density distribution of E. japonicus eggs. Although spatiotemporal factors were the main drivers of the spatiotemporal variation of the density distribution of E. japonicus eggs, these factors did not directly affect their distribution pattern. Rather, spatiotemporal factors indirectly affected the concentration distribution of E. japonicus eggs through SST, SSS, and depth. The optimal temperature range for the spawning of E. japonicus was wide, and the distribution of spawning grounds indicated a synergistic effect under low temperatures and low salinity and an inhibitory effect under high temperatures and low salinity. During the early spawning season in April, the central spawning ground was located in the deep waters southeast of Haiyang Island. Alongside continuously rising water temperatures, both the size of the spawning ground and the concentration distribution of E. japonicus eventually reached their annual peaks between late May to early June, thus, constituting the peak spawning season. During this period, the central spawning ground was located around Shicheng Island and the Yingna River estuary. From June, however, water salinity decreased along the southern coast of the Liaoning Peninsula due to increasing coastal freshwater runoff. The inhibiting effect of higher temperatures and lower salinity drove the spawning fish away from the coastal waters and into deeper offshore sea areas. The E. japonicus spawning grounds, thus, migrated further offshore with SSS as the dominant factor driving this migration. As autumn and winter arrived, the spawning activity of E. japonicus gradually ended. E. japonicus eggs were sparsely scattered in the investigated sea area during October. In November, large numbers of E. japonicus began gathering in the center of the investigated sea area and gradually migrated further southward to their wintering habitat. Accordingly, no E. japonicus eggs were collected during the month of December. The main spawning grounds of E. japonicus were located in the coastal waters of Shicheng Island and the Yingna Estuary, and gradually showed a contraction trend after July with the gradual weakening of E. japonicus spawning activities. Overall, from April to December 2021, the distribution of E. japonicus eggs in offshore waters of the Liaoning Province in the North Yellow Sea showed a migration trend from the southern offshore deep water area to the northern offshore shallow water area, and then to the southwestern offshore deep water area. This study elucidated the spatial and temporal distribution patterns of E. japonicus spawning grounds and its influencing factors, which indicated that the Tweedie-GAM method could be effectively applied to analyze the relationship between the early fish resources and environmental factors. This method showed good performance in solving 0-value problems. The results provide a scientific basis for the conservation and management of E. japonicus spawning grounds in the North Yellow Sea, assist with evaluation of the current situation and development trend of E. japonicus resources in the sea area, and provide an important reference for the rational development and utilization of E. japonicus resources and research on spawning ground protection strategies.

Abstract:The ocean is the largest carbon pool on earth and plays an important role in the global carbon cycle. Marine organisms contribute over half of the global carbon fixed by photosynthesis every year. Therefore, much attention has been paid to the role of the ocean in the carbon cycle and carbon sequestration. Macroalgae are an important component of coastal ecosystems, with strong and highly efficient carbon fixation capacity. The net primary productivity (NPP) of macroalgae was higher than that of phytoplankton and other primary producers in coastal waters. Parts of the photosynthates are released into the sea in the form of dissolved organic carbon (DOC) and particulate organic carbon (POC) during algae growth. Dissolved organic carbon and POC have attracted widespread attention because they can play important roles in carbon cycling and carbon sequestration in coastal ecosystems. However, the organic carbon release rate of macroalgae, its relationship with primary productivity, and the regulatory factors require further study. The growth of algae and the organic carbon release are influenced by temperature, light intensity, photoperiod, and other factors. However, organic carbon release rates among algae species might be quite different. Therefore, studying the regulatory effects of different factors on organic carbon release by algae and organic carbon release rate distribution has important implications for quantitatively evaluating the organic carbon release ability of algae and its contribution to the coastal ecosystem carbon cycle. In recent years, Sargassum horneri is the most common wild macroalgae in the Yellow Sea. Its ecological role in the coastal ecosystem has attracted widespread attention. Few studies have focused on organic carbon release by S. horneri. This study conducted an orthogonal experiment to measure DOC and POC release rates of S. horneri at different temperatures (5, 15, and 25 °C), light intensity [86, 172, and 258 μmol/(m2·s)] and photoperiod (L:D=6 h:18 h, L:D=12 h:12 h, L:D=24 h:0 h, L means light time, D means dark time) to study the organic carbon release rate of S. horneri, its relationship with NPP, and the main regulatory factors. The release rates of DOC were 0.653–4.785 mg/(g·h) and the release rates of POC were 0.066–0.322 mg/(g·h). There is an order of magnitude difference between DOC and POC. This indicated that DOC was the main form of organic carbon released by S. horneri. The highest DOC release rate [4.785 mg/(g·h)] occurred at 25 °C, 172 μmol/(m2·s) light intensity, and L:D = 6 h:18 h. The highest POC release rate [0.322 mg/(g·h)] occurred at 25 °C, 258 μmol/(m2·s) light intensity, and L:D = 24 h:0 h. The organic carbon release ability of S. horneri is consistent with previous studies of other macroalgae. This study found that changes in temperature and light conditions significantly affect the ability of S. horneri to release organic carbon. Temperature was the main factor affecting the release rate of DOC; the DOC release rate increases with increasing temperature. The DOC release rate initially increases, then decreases with increasing light intensity; it increases with a decrease in the photoperiod. Light intensity was the main factor influencing the POC release rate; increasing light intensity and photoperiod accelerated the POC release rate. However, the POC release rate slightly decreased when the light intensity increased from 172 to 258 μmol/(m2·s). There was a greater POC release rate under extreme temperature stress (5 and 25 °C). This study showed that a proportion of DOC and POC in NPP were unstable in macroalgae. The range of DOC/NPP was 4%–130%. POC/NPP was relatively concentrated to approximately 0.4%–5.9%. The NPP was significantly lower in the experimental group with a high ratio of organic carbon compared with other experimental groups. The DOC release rate negatively correlated with NPP, but the negative correlation was poor in the low temperature groups. There was no significant correlation between the POC release rate and NPP. POC/NPP was relatively stable [approximately (1±0.6)% when NPP > 9 mg C/(g·h)]. The growth pressure of S. horneri was high when NPP < 4 mg C/(g·h), and the regulatory effect of organic carbon was manifested as the release under environmental stress, with POC/NPP increasing to 4%–6%. This section was modified for clarity. Please check that your meaning was retained. This study provides scientific and technological support for an in-depth understanding of the contribution by macroalgae to the coastal ecosystem carbon cycle.

Abstract:To meet the growing demand for animal food among the population, China has proposed the strategy of building a “Blue Granary,” relying on marine space, marine biological resources, and the application of modern marine high-tech. Industrialized recirculating aquaculture (also known as land-based factory aquaculture, factory aquaculture, or industrial fish farming) has the advantages of high production efficiency and small land occupation. It is a high-density, high-yield, high investment, and cost-effective aquaculture method. The recirculating aquaculture system is in line with the “Blue Granary” strategy, effectively reducing water pollution while ensuring food security. Therefore, in recent years, recirculating aquaculture in China has developed rapidly. In this context to achieve intelligent regulation of the inflow velocity of industrial recirculating aquaculture, this article firstly summarizes the three main factors that affect the inflow velocity from previous research: The flow field velocity of the aquaculture pool, the discharge velocity of the bait (residual bait), and the velocity of temperature regulation. With the improvement of computer software and hardware, computational fluid dynamics (CFD) is gradually being applied in various fields. CFD provides cheap tools for simulation, design, and optimization, as well as tools for analyzing three-dimensional complex flows. In complex cases, measurements are often difficult, even impossible, and CFD can easily provide detailed information on all flow fields. Compared with conventional experiments, CFD has the advantages of no restrictions on parameters, lower cost, and no interference in the flow field. This method can be used in aquaculture to solve the problems of temperature control effects, solid particle emission efficiency, and flow zone division that cannot be directly measured in actual production under different flow rates. At present, there are few studies reporting CFD numerical simulation of multiphase flow in aquaculture. Existing research focuses on the impact of the inlet and outlet structure and the shape of aquaculture ponds on the flow field, and the effect of sewage collection. There are few reports on the use of numerical simulation methods to design the regulation scheme of inlet flow velocity. Because of the advantages of CFD, we chose it for simulation. Secondly, the specific model used in the numerical simulation was determined through experimental verification, research discussion, and other methods. Based on this, grid independence verification was conducted, indicating that the simulation results under the number of grids in the text are not affected by the number of grids, and the simulation results are reliable. Thereafter, based on the above research, and taking Scophthalmus maximus as an example, the effects of different inlet flow rates on the flow field, sewage discharge, and water temperature regulation in aquaculture ponds were simulated. The results showed that the inlet flow rate significantly affects these three factors. Therefore, the inlet flow rate can be adjusted according to production needs. Based on the simulation results, a set of inlet flow velocity control schemes were proposed: For the feeding stage, low flow velocity can be used to reduce feed costs (inlet flow velocity = 0.2 m/s); after eating, the feed can be quickly discharged at a high flow rate for a short period of time (inlet flow velocity = 1.2 m/s for 20 s); at abnormal water temperature, different flow rates and times can be used to adjust the water temperature in the breeding pool (water inlet temperature = 15 ℃; an inlet flow velocity of 1.2 m/s can be used to inject water for 230 s, reducing the water temperature in the breeding pool from 22 ℃ to 18 ℃). Numerical simulation experiments can be designed based on this method for different aquaculture environments and organisms to determine the inflow velocity control scheme. Unlike traditional methods, the numerical simulation method proposed in this study for regulating the inflow velocity of recirculating aquaculture systems can be used to determine the inflow velocity control scheme at a lower cost. The method can directly measure the discharge of particulate matter, the overall water temperature, and the flow field in aquaculture ponds. This method is safer than the method of indirectly measuring water quality to determine the inflow velocity control scheme. In terms of regulation time, traditional methods are only accurate to the hour in regulating the inlet flow rate, whereas this method is accurate to the second, which makes it more reliable to determine the regulation of the inlet flow rate. Therefore, from the perspectives of cost, safety, and accuracy, it is better to use numerical simulation methods to regulate the inflow velocity of recirculating aquaculture systems. In actual production, the method described in this study can be used to determine the inflow velocity control scheme, which can be combined with the control system to achieve automatic control, reduce breeding costs, and increase breeding success.

Abstract:The total saline-alkaline land area in China is about 99.13 million hectares distributed across northern China, coastal areas, and areas along the bank of the Huanghe River. About 46 million hectares of saline-alkaline water areas are distributed around these saline-alkaline lands, most of which are thalassic and characterized by a high pH value in excess of 8.8 associated with high-carbonate alkalinity concentrations and various types of ion imbalances. Saline-alkaline waters are stressful environments in which only relatively few organisms are able to survive. Consequently, most of the saline-alkaline water resources have been desolate for a long time. The effective utilization of saline-alkaline water resources will benefit restoration of saline-alkaline habitats and the expansion of aquaculture space. Naked carp (Gymnocypris przewalskii) are endemic to the austere saline-alkaline environment of Qinghai Lake. Due to overfishing in the 1960s and environmental changes in the lake area, the resources necessary for naked carp survival in Qinghai Lake declined substantially. At present, the major measures to protect the naked carp and maintain the ecological balance of Qinghai Lake are through a fishing ban and artificial stocking and releasing. The feeding behavior of fish under natural conditions has obvious rhythm characteristics, which is an important research topic for healthy aquaculture. To explore the characteristics of self-feeding rhythm and growth performance of fish in a saline-alkaline environment and provide basic data for the protection of native saline-alkaline fish, naked carp were taken as representative in this study. First, the freshwater and lake water group with natural photoperiod (14L:10D) and the lake water group with darkness (24D) were set. The artificial lake water was prepared according to the ionic composition of Qinghai Lake, with the contents of Na+ 23.05%, K+ 1.34%, Ca2+ 0.11%, Mg2+ 6.88%, HCO3– 7.09%, CO32– 5.07%, Cl– 40.39%, and SO42– 16.07%. The measured salinity of the artificial lake water was 15.08, and the carbonate alkalinity was 27.53 mmol/L. According to the local photoperiod of Qinghai Province, the whole day was divided into five periods as 05:00–08:00, 8:00–11:00, 11:00–15:00, 15:00–19:00, and 19:00–05:00. The feeding rhythm experiment lasted for 5 d, and the average food intake of each period was calculated. The results showed that naked carp had an obvious daily feeding rhythm during their natural photoperiod. In the natural photoperiod, the feeding peak was from 08:00 to 11:00, and the low feeding period was from 05:00 to 08:00 in freshwater. In the lake water, naked carp showed high and continuous feeding from 08:00 to 19:00, and their average hourly feed intake was significantly higher than that from 05:00 to 08:00 and 19:00 to 05:00. Therefore, naked carp were determined to be the daytime feeding fish type. In addition, the high proportion and the continuous feeding in daytime in lake water indicated that the osmotic and acid-base regulation of naked carp in saline-alkaline water may enhance their diurnal feeding rhythm. Whereas in the continuous dark environment, the feeding rhythm of naked carp was weakened, and the average hourly food intake of each period was similar. To explore the growth performance of naked carp under a self-feeding rhythm, the lake water group and the freshwater control group with natural photoperiod were set up. After 63 days of self-feeding, the individual body length and weight of the naked carp were measured after being anesthetized with MS-222. The length growth rate (1.19±0.17)%, weight growth rate (10.66±0.98)%, and specific growth rate (0.16±0.02)%/d of naked carp in the lake water group were significantly lower than those in the freshwater group [length growth rate (18.66±0.41)%, weight growth rate (67.32±3.05)%, and specific growth rate (0.82±0.03)%/d)], indicating that the growth of naked carp was inhibited by high salinity and carbonate alkalinity environment. The parameter b of body length-weight relationship curve of the naked carp in both the lake water group and the freshwater group was less than three, which showed that the naked carp was a negative allometric growth fish. The b value of the lake water group was lower than that of the freshwater group, and the body length of naked carp increased faster than body weight in the lake water. The growth characteristics of naked carp were affected by the high saline-alkaline environment. The self-feeding rhythm and growth performance of naked carp provided a basic knowledge for creating a feeding strategy for fish cultured in a saline-alkaline environment and recovering endangered native saline-alkaline fish.

Abstract:The complete intestinal structure is important to ensure the rapid and h ealthy growth of fish. However, the feed composition, aquaculture water environment, intestinal microbial population, and other factors may affect the intestinal health of fish. Intestinal health problems caused by feed ingredients are mainly due to the antinutrient factors contained in raw materials. Antinutritional factors contained in high-level soybean meal can cause oxidative damage to the intestine, thus, inducing soybean meal-induced enteritis (SBMIE), which leads to a decreased appetite and the slow growth of fish. Alleviating the damage of soybean meal to the fish intestinal tract and improving intestinal health through nutrition are essential methods for ensuring the sustainable development of the feed industry, which has significant ecological and economic significance. As a functional amino acid, arginine is a precursor to the synthesis of bioactive substances, such as urea, glutamic acid, creatine, proline, polyamine, and nitric oxide. Arginine modulates metabolic regulation, including growth, immunity, intestinal barrier, and endocrine regulation. It plays a vital role in the immune regulation, maintenance, and protection of the intestinal mucosal structure and function. It has been reported that arginine is beneficial for repairing intestinal mucosal injury in poultry and aquatic animals. In this study, the carnivorous marine economic fish Sebastes schlegelii (54.97±0.12) g were used to investigate the repair effect and mechanism of arginine on SBMIE. This study aimed to provide a scientific basis for the application of arginine for maintaining the intestinal health of fish and provide a reference for the application of plant protein to the compound feed of the carnivorous economic fish S. schlegelii. The purpose of this study was to investigate the repairing effects of arginine on the growth performance, arginine metabolism, intestinal structure, antioxidant performance, relative expression levels of intestinal tight junction protein genes (occludin, clnd15, and zo-1), and inflammatory factor-related genes (il-1β, il-8, il-15, and tlr8) and anti-inflammatory factor-related gene (il-12b) of S. schlegelii with SBMIE. S. schlegelii were fed high-level soybean meal (40%) for 28 days to induce SBMIE. SBMIE-S. schlegelii weighing (54.97±0.12) g were used as the study animals. Four isonitrogen and isoenergetic experimental feeds were formulated. The basic formula was supplemented with 30% soybean meal, arginine 0 supplementation as the control group (D0), and 1%, 2%, and 3% arginine supplementation as the treatment groups, named D1, D2, and D3, respectively. Each diet group had three replicates, and each replicate consisted of 40 fish. The fish were randomly placed in 12 homemade cages (60 cm × 60 cm × 90 cm). The experiment lasted for 6 weeks. The experimental fish were fed twice a day (08:00 and 17:00), with the initial feeding amount being 1% of the body weight, and the feeding amount being adjusted according to the feeding situation. During the experiment, the bottom of the cages was cleaned, and the water was changed every day to maintain the water temperature at 18~22 ℃, the dissolved oxygen at > 6 mg/L, the pH at 7.6~8.2, the ammonia nitrogen content at < 0.05 mg/L, and the nitrite nitrogen content at < 0.05 mg/L. The light cycle was the natural cycle. The results showed that the weight gain rate of the fish in the D2 and D3 groups was significantly higher than that in D0 group (P<0.05). The hepatosomatic and viscerosomatic indexes of the fish in the arginine treatment groups were significantly lower than those in the D0 group, and the condition factor was significantly higher than that in the D0 group (P<0.05). There was no significant effect on the survival rate (P>0.05). Diamine oxidase (DAO) activity, NO content, and iNOS activity values in the serum of the treatment groups were significantly lower than those in the D0 group (P<0.05). The serum T-NOS activity in the D2 and D3 groups was significantly lower than that in the D0 group (P<0.05). The duplicature height in the treatment groups was significantly higher than that in the D0 group, while no significant difference was found in the duplicature number and muscle thickness (P>0.05). In group D0, the intestinal mucosa lamina propria widened, and the number of goblet cells increased, while in groups supplemented with arginine, the intestinal mucosa was intact, and the problems mentioned above improved significantly. Intestinal total antioxidant capacity (T-AOC) in arginine supplementation groups was significantly increased, and the highest value was found in group D2 (P<0.05). The malondialdehyde content in groups D2 and D3 was decreased significantly compared to that in the D0 group (P<0.05). The relative expression of occludin mRNA in each treatment group was significantly upregulated compared to that in the D0 group (P<0.05). The relative expression level of clnd15 mRNA in the D2 group was significantly higher than that in the D0 and D1 groups (P<0.05). The relative expression level of zo-1 mRNA in group D1 was significantly higher than that in the other groups (P<0.05). The relative expression levels of IL-1β, IL-15, and TLR8 mRNA were downregulated in all treatment groups, while the relative expression of IL-12b mRNA was upregulated (P<0.05). No significant differences were found in IL-8 mRNA relative expression (P>0.05). In conclusion, under the conditions of this experiment, the growth and antioxidant performances of S. schlegelii with SBMIE were significantly increased, arginine metabolism and the intestinal structure were improved significantly, and the relative expression of intestinal tight junction protein and anti-inflammatory factor-related genes was upregulated, while that of inflammatory factor-related genes was downregulated, with arginine supplementation in a high-level soybean meal diet. Arginine (2% best) was effective in repairing SBMIE of S. schlegelii. The results of this study provide a theoretical basis for the mechanism of repairing SBMIE with arginine.

Abstract:Coregonus ussurinsis Berg is a rare cold-water fish found in Heilongjiang Province, which has high nutritional and economical value. The growth traits of fish are critical breeding target traits, and improving the growth efficiency of cultured fishes has always been a major issue for researchers. As an endangered fish, very limited research has been conducted on C. ussurinsis, and studies on its growth and development are still lacking. Therefore, investigating gene expression in C. ussurinsis muscles would significantly contribute to our understanding of their muscle development. RNA-Seq was used to find and study the specific genes and pathways of muscle development under different conditions. Recently, transcriptome sequencing has been applied to diverse animal populations, aiding in the selection of candidate genes related to important traits by comparing the global gene expression profiles between different animal populations with specific characteristics. This study aims to understand the genetic basis of muscle development in C. ussurinsis at the transcriptome level and to provide new insights into growth and development. To explore the molecular regulation mechanism of growth traits of C. ussurinsis, F2 individuals of C. ussurinsis were randomly selected from the mixed pool for test grouping (fast-growing group and slow-growing group). The dorsal muscle tissue was clipped from 10 fast-growing individuals (219.20±38.66 g, weight) and 10 slow-growing individuals (74.30±17.86 g, weight) for transcriptome sequencing to construct six cDNA libraries. High-throughput sequencing from Illumina NovaSeq 6000 and bioinformatics was used to determine the abundances and characteristics of transcripts. The differentially expressed genes were screened with FDR (false discovery rate)<0.05 and |log2FoldChange|>1; the functions of these differentially expressed genes (DEGs) were annotated and analyzed by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify the genes and genetic pathways related to the development of muscle in C. ussurinsis. Moreover, to verify the sequencing results, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression levels of DEGs. The results showed that the correlation coefficients of all the samples used for transcriptome sequencing were above 0.83, indicating high correlation between the samples and experimental reliability. Transcriptome sequencing results showed that a total of 295 605 738 raw reads were assembled from the six cDNA libraries, and 283 133 612 clean reads were obtained after quality control. Q20 and Q30 sequences accounted for above 97.80% and 93.90%, respectively, and the content of GC bases accounted for more than 49.1% of the total bases. Through comparison with the genome using EdgeR software, which was used to analyze the differences in gene expression, 2 211 DEGs were preliminarily obtained from muscle, including 659 novel genes. Compared with the slow-growing group, 583 differential genes were up-regulated, and 1 628 differential genes were down-regulated in the fast-growing group. Function enrichment analysis found that the DEGs participated in 3 620 GO terms. Among them, 2 457 biological processes were primarily involved in cellular and metabolic processes; there were 782 molecular functions, primarily involved in binding function and catalytic activity processes, and 381 cellular components, primarily involved in cell and cell component processes. The enrichment analysis of the KEGG pathway found that a total of 251 signal pathways were obtained, among which 73 were significantly enriched (P<0.05). Among them, the up-regulated DEGs were mainly involved in glycolysis/gluconeogenesis, methane metabolism, and biosynthesis of amino acids, while the down-regulated DEGs were mainly involved in osteoclast differentiation, IL-17 signaling pathway, and biosynthesis of amino acids. The genes related to muscle growth were significantly (P<0.05) enriched in the MAPK signaling pathway, PI3K-Akt signaling pathway, tight junction, insulin signaling pathway, glycolysis/gluconeogenesis, and PPAR signaling pathway. These pathways might be closely related to muscle growth. Combined with the GO functional annotation, the KEGG pathway enrichment, and the annotation results, 31 potential growth-related candidate genes were preliminarily screened. Protein-protein interaction networks were used to further analyze the relationship between these differential genes. It was found that atp2a2, atp2a1, g6pc, igfbp1, myh1, myh4, myh6, myh7, myh9, and myh13 might be closely related to muscle growth regulation, and these 10 genes can be used as crucial candidate genes for the growth regulation of C. ussurinsis. The qRT-PCR validation of 10 randomly selected differential genes showed consistent gene expression trends with the transcriptome sequencing results, which indicated that the results obtained by transcriptome sequencing in this study were accurate. A total of 10 growth-related essential candidate genes were screened in this study; these genes affect the growth of C. ussurinsis by regulating their expression levels in muscle tissue. These results provide vital information for the further understanding of the molecular basis and marker-assisted breeding of the growth regulation of C. ussurinsis.

Abstract:Clock genes play a pivotal role in the rhythm maturation of bony fish ovaries. To explore the role of clock genes in the rhythmic ovarian development and maturation of tongue sole (Cynoglossus semilaevis), we used real-time fluorescent quantitative PCR technique to analyze the expression profiles of circadian locomotor output cycles Kaput 1a (Clock1a), brain and muscle Arntlike 1a (Bmal1a), Cryptochrome 1a (Cry1a), Cryptochrome 2 (Cry2), and Period 2 (Per2) at the ovarian stages Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ. The coding DNA sequence (CDS) of five clock genes were cloned and phylogenetically analyzed. We found that the CDS sequence length of Clock1a was 1 620 bp and encoded 539 amino acids, with the encoded amino acid sequence of Clock1a having a predicted molecular weight of 81.9 kDa. Clock1a has the functional domain PASD1 (consisting of 64 amino acids) and PAS11 (consisting of 103 amino acids). The CDS sequence length of Bmal1a was 1 881 bp and encoded 626 amino acids, with the encoded amino acid sequence of Bmal1a having a predicted molecular weight of 68.9 kDa. In the Bmal1a sequence, the functional domain PASD3 is composed of 63 amino acids, and the functional domain PAS11 is composed of 103 amino acids. The CDS sequence length of Cry1a was 1 896 bp and encoded 631 amino acids, with the encoded amino acid sequence of Cry1a having a predicted molecular weight of 71.4 kDa. Its functional domain FAD7 was composed of 199 amino acids. The CDS sequence length of Cry2 was 2 007 bp and encoded 669 amino acids, with the encoded amino acid sequence of Cry2 having a predicted molecular weight of 76.0 kDa. The Cry2 sequence contains a functional domain FAD7 consisting of 199 amino acids. The CDS sequence length of Per2 was 4 248 bp, encoding 1 415 amino acids, and the encoded amino acid sequence of Per2 has a predicted molecular weight of 154.0 kDa. In the Per2 sequence, the functional domain PeriodC consists of 295 amino acids and a functional domain PAS11 consists of 102 amino acids. The neighbor-joining method was used to analyze the Clock1a, Bmal1a, Cry1a, Cry2, and Per2 phylogenetic relationships between C. semilaevis and other bony fish, amphibians, birds, and mammals. The homology of Clock1a, Bmal1a, Cry1a, Cry2, and Per2 with other bony fish was 60%–79%, 94%–100%, 85%–91%, 84%–94%, and 70%–84%, respectively. Therefore, we believe that these five amino acid sequences show strong conserved property. In addition, the homology of Cry1a and Cry2 was 62%, indicating that Cry1a and Cry2 evolved differently during the evolution of C. semilaevis. In the constructed phylogenetic tree, Clock1a, Bmal1a, Cry1a, Cry2, and Per2 of C. semilaevis were clustered together with other bony fishes, indicating a close relationship between C. semilaevis and other bony fish in the evolutionary process. Moreover, the homology of Clock1a between C. semilaevis and mammals, birds, and amphibians is low, indicating that there are evolutionary differences in the evolutionary process of Clock1a. The high homology of Bmal1a, Cry1a, Cry2, and Per2 with mammals, birds, and amphibians suggests that these four clock genes were strongly conserved during the evolution of C. semilaevis. In the present study, we found that the expression levels of five clock genes were high in stagesⅡ and Ⅲ, which were equivalent to the non-reproductive season (P<0.05). However, the expression levels of five clock genes were low in stages Ⅳ and Ⅴ, which were equivalent to the reproductive season (P<0.05). Therefore, the expression profiles of Clock1a, Bmal1a, Cry1a, Cry2, and Per2 in the ovaries of C. semilaevis also have seasonal characteristics. The ovarian development and maturation of C. semilaevis goes through stages Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ, and then reaches stage Ⅱ again and starts a new reproductive cycle. The variation patterns of seasonal factors, such as light and temperature, in fish ovaries were consistent with the annual expression patterns of the five clock genes in this study. Therefore, it can be considered that the expression of clock genes in C. semilaevis has an annual cycle. The findings presented in this study can enrich the theory of ovarian development and maturation of C. semilaevis and provide a theoretical basis for improving breeding technology and seedling efficiency.

Abstract:Color pattern plays a vital role in animal survival and communication. The type, distribution, and pigment state of pigment cells, and the reflective ability of iridophores determines body color. It varies adaptively in response to external environmental changes and physiological states. The skin pigmentation pattern reflects the number and arrangement of chromatophores. Some fish with rich color patterns, including egg spot patterns, blotch patterns, melanism, horizontal stripe patterns, and vertical bar patterns, have been studied increasingly. This study observed the formation, distribution, and main pattern of chromatophores in 1–60-day-old Misgurnus anguillicaudatus after hatching. Larval melanocytes were first observed in the yolk sac of loach larvae at 3 h post-hatching. From the larval to juvenile stage at 21 days, larval melanocytes appeared on the loach body surface. From the juvenile stage at 22 days to the adult stage, adult melanocytes appeared on the loach body surface. Iridocytes were first observed in the eyes of one-day-old larvae but not on the body surface until they were 12 days old. Xanthophores appeared on the body surface of seven-day-old juveniles. At 2–21 days post-hatching, the melanocytes in the loaches were larval, and their shape changed from star- to snowflake-shaped before forming a black spot. From 22 days, different morphological adult melanocytes formed on the body surface of the loaches with three types of black spots. Chrysanthemum-shaped melanocytes regularly aggregated into large black spots on large black spot loaches. Round and dendritic melanocytes gathered to form small black spots on small black spot loaches. Dendritic melanocytes were evenly distributed on non-black spot loaches. The pigmentation-related mitfa gene was obtained from M. anguillicaudatus using the rapid amplification of cDNA ends (RACE) approach with the SMARTer RACE 5´/3´ Kit User Manual according to the manufacturer’s recommendations and was analyzed using bioinformatics and quantitative methods. The results showed that the mitfa gene encoded a protein with 408 amino acids with a calculated molecular mass of 45.68 kDa and an estimated isoelectric point of 7.16. MITFa contained MITF_TFEB_C_3_N, bHLH-Zip, and DUF 3371 domains. MITFa was well-conserved compared to MITF of various species with a higher degree of sequence similarity with other fishes (58.8%–83.2%). The qRT-PCR results showed that the mitfa mRNA was expressed at all stages of embryonic development and reached a peak value at the fertilization stage. mitfa expression was detected in all examined tissues of the three types of loaches, and the highest level of expression was detected in both muscle and dorsal skin (P<0.01). This study explored pigmentation formation and mitfa expression, serving as a foundation for gaining further insight into the genetic mechanism of body color formation in M. anguillicaudatus.

Abstract:As a benzoylurea insecticide, diflubenzuron (DFB) has been widely used in the prevention and control of diseases and insect pests in vegetables, fruits, grain cultivation, and other fields in recent years. In addition, because of its low acute toxicity, excellent biological activity, and specific action mechanism, DFB has been widely used in aquaculture to kill bacteria and lice. With its large-scale production and widespread use, its partial residue remains in environmental media, such as water, soil, and the atmosphere, and causes direct or indirect chronic toxicity to aquatic organisms. The residual DFB enters the human body along the food chain and poses a threat to human health when it reaches a certain concentration through chronic exposure and long-term accumulation. At present, research on DFB insecticides has been limited to the usage and dosage, residual metabolism, and its toxicological effects on the environment. There have been no reports, to our knowledge, on the effect of DFB on gene expression in carp liver in China. The purpose of this study was to explore the differential expression of genes in carp liver under DFB stress. In this study, carp (Cyprinus carpio) was selected as the research subject, and three exposure experiments for 15 days under 0.1 and 1.0 mg/L medicated bath concentrations were carried out in parallel for each concentration. High-throughput sequencing of the liver was performed using the Illumina NovaSeq 6000. Differentially expressed genes (DEGs) were screened by Padj < 0.05 and |log2FoldChange| ≥1, and bioinformatics analysis, such as Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, were performed. The transcriptome analysis results showed that 2 406 and 2 688 DEGs changed significantly at 0.1 and 1.0 mg/L exposure concentrations, respectively, and 821 DEGs were co-expressed between the two groups. GO analysis results showed that DEGs in the DFB exposure group were enriched in biological processes, cell compositions, and molecular functions. KEGG enrichment analysis showed that DEGs in the low DFB concentration exposure group were significantly enriched in metabolic pathways, such as biodegradation and metabolism of xenobiotics, lipid metabolism, carbohydrate metabolism, amino acid metabolism, signal molecules and interactions, endocrine system, and immune system. In addition to the above metabolic pathways, DEGs in the high DFB concentration exposure group were significantly enriched in metabolic pathways, such as folding, classification and degradation, transport, and catabolism. Studies have shown that DFB exposure causes the biodegradation and metabolism of xenobiotics, lipid metabolism, carbohydrate metabolism, and amino acid metabolism, and generates endoplasmic reticulum stress, inflammatory response, and immune toxicity. In summary, the results of this study provide basic data and a theoretical basis for further research on the molecular mechanism of DFB stress in carp.

Abstract:This study performed rational feeding of fermented feed to channel catfish and determined its effects on growth, the intestinal bacterial community, and metabolomics of channel catfish. There were three groups: continuous feeding (A, continuous addition of fermented feed to the puffed feed), interval feeding involving weekly intervals addition of fermented feed added to the puffed feed (B), and the control group only fed the puffed feed (C). The experiment lasted 6 months. Channel catfish were weighed and the intestinal flora and metabolomics were detected by 16S rRNA sequencing technology and liquid chromatography-based metabolomics technology, respectively after the experiment. The final body weight (FBW) of channel catfish was significantly higher in the interval feeding group than that in the control group and the continuous feeding group (P<0.05). The richness and microbial diversity of the intestine was the highest in the interval feeding group, while the richness of the continuous feeding group was the lowest (P>0.05). Firmicutes, Actinobacteriota, Cyanobacteria, Proteobacteria, Fusobacteriota, Chloroflexi, and Deinococcota were the dominant bacterial phyla among the intestinal flora. The dominant intestinal genera included norank_f_norank_o_Chloroplast, Mycobacterium, Cetobacterium, Romboutsia, Exiguobacterium and Clostridium_sensu_stricto_1. Metabolomic analysis showed that the continuous feeding group mainly affected galactose metabolism and the phosphotransferase system of the intestinal flora in channel catfish through the significant upregulation of N-acetyl-D-galactosamine (P<0.05) that affected the digestion and absorption of carbohydrates by fish. The differential metabolites L-serine and L-phenylalanine were significantly upregulated (P<0.05) in the interval feeding group. This affected sulfur metabolism and amino acid metabolism of the intestinal flora, and affected energy absorption, anti-inflammation, and immunity of the channel catfish. This study provided a theoretical basis for the exploration of feeding methods using fermentation feed and healthy green breeding of channel catfish.

Abstract:Grouper, a species of coral reef fish, exhibits a wide geographical distribution within the warm waters of the tropical and subtropical regions across the globe, primarily inhabiting the middle and lower layers of water. Characterized as a substantial marine economic fish, grouper possesses considerable nutritional value, boasts a high market worth, and garners significant consumer demand. Its popularity among consumers is attributed to its inherent attributes, and it holds immense potential for further cultivation and breeding endeavors. This study utilized micro-satellite (MISA) software to investigate the distribution characteristics of microsatellites in the genomes of five grouper species (Epinephelus akaara, E. coioides, E. fuscoguttatus, E. lanceolatus, and E. moara). A custom script was developed to analyze the screening results, and statistical analyses were conducted on the microsatellite repeat types, duplicate copy types, and core copy numbers in the genomes of the five grouper species. Over 280 000 microsatellite sites were identified from the entire genomes of the five grouper species. The relative abundance of microsatellites ranged from 271–296, with a total length ranging from 6.30–7.06 Mb. The average length of the microsatellites was approximately 22 bp, and their proportion in the genomes ranged from 0.59%–0.67%. These results provide insights into the distribution characteristics of microsatellites in the genomes of these five grouper species and can inform future studies on their genomic architecture and evolution. The repetitive types of microsatellites were analyzed in terms of number, proportion, and relative abundance. The number, proportion, and relative abundance of repetitive types followed a consistent pattern, with the highest number of double base repeats, followed by single base repeats. This pattern decreased as the number of repeat units increased. A, AC, AAT, AAG, AGC, AATC, AAAT, AGAT, AATG, AGAGG, AAAAT, AAGAT, ACAGAG, AAANNN, and AANNNN (N represents any of the three bases except A) were the most dominant types of each duplicate copy type. Type A accounted for 90.00% of single base repeats, while type AC was the most dominant in double base repeats, accounting for nearly 80.00%. Interestingly, the content of the CG duplication category was the least, accounting for only 0.04%–0.10% in the five grouper species. This may be owing to the fact that the composition content of the four bases in the different species' genomes is different, and there may be structural problems with different bases. The results of this study provide insight into the distribution characteristics of microsatellites in the genomes of these five grouper species. The high frequency distribution of AGG and AGC in the dominant types of triple base repeats may play a crucial role in regulating genes involved in immunity, disease, and other genes in groupers. Previous studies show that AGG is a well-known binding site for numerous transcription factors involved in early growth and development of various species. Additionally, the change of base repeat polymorphism of the AGC category is directly linked to genetic diseases and holds significant evolutionary and medical research value. AAAN, AAAAN, and AAAAAN are dominant repeat types that are widely distributed in mammals among the four, five, and six base repeat types, respectively. Different types of microsatellites show significant variability in the number of core copy numbers. Nevertheless, the number of duplicate copies of each type of microsatellite exhibit a consistent trend in the five groupers, and the number of microsatellites decrease with an increase in the number of duplicate copies. The analysis of microsatellite distribution revealed several key findings. First, over 95% of single base repeat copies were concentrated in a range of 12 to 25 times. The main number of copies for two base repeats ranged from 6 to 32 times, with a small peak between 11 and 14 copies, and decreasing numbers with increasing copies. The number of copies for four and five base repeats was mainly concentrated in the ranges of 5–16 and 5–17, and 5–14, respectively. Notably, AGAT, AAAG, AAGAG, AATAT, and AGAGG repeats exhibited a large number of copies, even when the number of copies was high. The increase in copy number may represent changes in polymorphism at these loci that may lead to disease or changes in corresponding functions. Overall, these findings provide important insights into the distribution and potential functional significance of microsatellites in the genome of the studied species. The distribution characteristics of microsatellites in the genomes of the five groupers provide a valuable basis to understand the evolutionary mechanisms and functional expression of these species. The distribution of duplicate copy numbers of each type of duplication displays two peaks at 6 and 12 repetitions, with the number of microsatellites decreasing with increasing numbers of core copies. Some duplication types show particularly prominent numbers in specific species, such as T, TA, and AGACAG in E. lanceolatus, copied 502, 803, and 48 times respectively; GAG, CACT, and CCACA in E. moara, copied 205, 652, and 111 times respectively. These variations highlight the importance of exploring the role of microsatellite loci to develop a better understanding of the genetic distance and kinship among the five groupers. This analysis lays the groundwork to develop high-quality microsatellite molecular markers, and facilitates the selection of favorable varieties and the development of new varieties. In general, these research results provide important data to understand the genomic characteristics of the five groupers and helps to conduct advanced genetic research on these species.

Abstract:Penaeus vannamei Boone is an important economic species in aquaculture. It has become the main species of shrimp cultured in China due to its fast growth rate, high yield, and delicious meat. Segmented culture, which is important for promoting the environmental adaptation of shrimp, reducing the negative impact of diseases and improving the success of culture, is considered one of the major methods of shrimp farming. The intermediate cultivation stage is an important stage of shrimp farming as it determines the success or failure of economic benefits. P. vannamei are usually reared in intensive aquaculture systems that are highly dependent on bait feeding, which makes optimizing feeding rates fundamental to obtaining farming benefits. The feeding rate is the amount of bait as a percentage of the body mass of the cultured object, which is an important factor affecting the shrimp factory farming. Feeding rate directly affects the survival and specific growth rate of the cultured species; an excessively slow feeding rate will slow the growth of cultured objects and affect the normal development, while an excessively high feeding rate will increase the cost of breeding and cause accumulation of residual bait, which reduces water quality and may even result in death of the cultured objects. Therefore, it is necessary to determine the appropriate feeding rate to improve baiting efficiency and to satisfy the needs of sustainable aquaculture. However, in the intermediate cultivation stage, the determination of feeding rates relies on farming experience and lacks a scientific basis. To the best of our knowledge, few scholars have analyzed the influencing mechanisms of different feeding rates on shrimp growth, physiology, and water environment separately from the perspectives of exogenous and endogenous factors; however, there is a lack of comprehensive studies on the combined effects of feeding rates. Therefore, there is an urgent need to elaborate on the intrinsic connections between different parameters, such as the growth and physiological indicators of P. vannamei, water quality, as well as the microbial communities in the culture environment. The main objectives of this study are (1) to analyze the effects of different feeding rates on water quality conditions, microbial community structure, non-specific immunity indicators, and the growth performance of shrimp during the intermediate cultivational stage; (2) to elucidate the interaction between feeding rates and different indicators; and (3) to determine the appropriate feeding rate of the intermediate cultivation of P. vannamei. In this study, three groups of feeding rates, namely T5 (5%), T7.5 (7.5%), and T10 (10%), were used to feed P. vannamei during the intermediate cultivation stage for 30 d. The effects of different feeding rates on water quality, microbial community structure, non-specific immune indices, and growth performance in the intermediate cultivation of P. vannamei were analyzed. During the experiment, water quality and shrimp growth were regularly tested, and pH, salinity, temperature, and dissolved oxygen were maintained in a range suitable for shrimp growth. At the end of the experiment, the hepatopancreas of shrimp was used for non-specific immunity activity testing, and the microbial community characteristics of different culture densities were analyzed using high-throughput sequencing technology (Illumina MiSeq). Results showed that the concentrations of total ammonia nitrogen (TAN), NO2–-N, and chemical oxygen demand (COD) gradually increased as the experiment continued, and significant difference could be observed at the end of the experiment, T10 > T7.5 > T5 (P<0.05). Analysis of the microbial community structure showed an increasing tendency in richness and diversity with increasing feeding rates. At the phylum level, Proteobacteria (50.36%–67.53%) and Bacteroldota (12.09%–67.53%) were dominant in all samples; at the genus level, the relative abundance of Vibrio (harmful to P. vannamei) was the highest at T10 (37.33%) and lowest at T5 (0.13%), while the relative abundance of Pseudoalteromonas (beneficial to P. vannamei) was the highest at T7.5 (9.78%) and lowest at T10 (0.28%). Results of the redundancy analysis showed that the microbial community in the culture water with a feeding rate of 5% was positively correlated with salinity and temperature, and salinity had greater effect on microbial communities than temperature. The microbial community with feeding rate of 7.5% and 10% was positively affected by TAN, COD, and pH, and the effect of TAN on microbial community was greater than that of COD and pH. The superoxide dismutase, catalase, alkaline phosphatase, and acid phosphatase activities were the highest in T7.5 and were the lowest in T10 (P<0.05). The final average weight and length of T7.5 and T10 were significantly greater than those of T5 (P<0.05), while there was no significant difference between T7.5 and T10 (P>0.05). Survival rate was the highest at T5 (83.12%) and lowest at T10 (68.52%), with a significant difference (P<0.05). Factor analysis showed that the highest overall score of 0.92 was achieved at T7.5. This study showed that a feeding rate of 7.5% could improve growth performance and non-specific immunity in P. vannamei, and T7.5 was recommended for the intermediate cultivation of P. vannamei. This study elucidated the interactions between feeding rates and different indicators and determined the appropriate feeding rate for the intermediate cultivation of P. vannamei. This study might help optimize the feeding rates for the intermediate cultivational stage with a view to providing technical guidance for the improvement of the success rate of P. vannamei.

Abstract:Acute hepatopancreatic necrosis disease (AHPND) is widely prevalent, has a rapid onset, and has high mortality in shrimp culture, making it a key limiting factor affecting shrimp aquaculture development in recent years, resulting in massive economic losses to the industry worldwide. Systematic studies that investigate which factors significantly correlate with the occurrence of AHPND, and further establishment of a prediction model for the occurrence of shrimp AHPND, are important for preventing and controlling the disease. In this study, Penaeus vannamei in pond culture were preliminarily analyzed for the coupling relationship between the occurrence and prevalence of AHPND in shrimps and pathogens, and for environmental and host autoimmune factors by assessing the environmental factors, pathogen abundance, and host health indicators under AHPND incidence. Then, a mathematical early warning model of AHPND occurrence in pond-cultured P. vannamei was constructed using Deep Forest algorithm. The occurrence of AHPND and its environment, pathogen, and shrimp immunity factors in pond-cultured P. vannamei were continuously monitored to explore the relationship between the occurrences of AHPND in relation to these factors. A total of 18 parameters were assessed, including the air and water temperature, salinity, pH, dissolved oxygen (DO), ammonia nitrogen (NH4+-N) and nitrite (NO2-N) concentrations, culturable bacteria and Vibrio in water, culturable bacteria and Vibrio in the shrimp hepatopancreas, the proportion of Vibrio in water and the shrimp hepatopancreas, and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM), and phenol oxidase (PO) in shrimp muscles. The parameter simulation prediction data based on the P. vannamei AHPND occurrence-related factor sequence (environmental factor, microbial factor, and shrimp health indicator) were constructed for the first time. The one-dimensional sequence was mapped into the three-dimensional space, different kernel functions were selected in combination with the actual classification problem to compare the model fitting accuracy, and the test algorithm optimized the parameters in the model. A total of 140 relevant data groups were collected under the same mode, and the groups of additional exogenous inputs during the breeding process were eliminated. After deleting invalid data, there were 100 groups of classified monitoring data, including 25 groups of morbidity data and 75 groups of health data. Moreover, the model was affected due to the dimensional and quantitative differences among different factors. In order to improve the speed of subsequent experimental training and prediction accuracy, the 100 groups of training test data processed by the mapminmax function were normalized for data processing. The relationship between 18 parameters and the occurrence of AHPND in P. vannamei was analyzed using Pearson´s correlation, and the main influencing factors were further screened using pairwise analysis between the factors. Pearson´s correlation analysis indicated that the incidence of AHPND positively correlated (P<0.05) with salinity, the number of culturable bacteria and Vibrio in the shrimp, the proportion of Vibrio in the shrimp, the number of culturable bacteria and Vibrio in water, and the activities of LZM, ACP, and PO in shrimp muscles. The correlation coefficients were 0.350 1, 0.574 1, 0.521 1, 0.391 1, 0.374 7, 0.238 3, 0.438 2, 0.257 1, and 0.228 9, respectively, indicating that AHPND was more likely to occur with an increase of these parameter values within a certain range. The incidence of AHPND negatively correlated with water temperature (P<0.05), and the correlation coefficient was –0.227 9. Moreover, the water temperature, pH, DO, NH4+-N and NO2-N concentrations, Vibrio proportion in water, AKP, and SOD had a weak correlation with the incidence of AHPND (P>0.05). Furthermore, parameters were removed in the model construction process according to the correlation between parameters and factors. The occurrence of AHPND in P. vannamei directly and significantly correlated with seven parameters, including the total number of shrimp bacteria, the total number of shrimp Vibrio, LZM, the proportion of shrimp Vibrio, the total number of water bacteria, salinity, and the total number of water Vibrio. The prediction performance of three popular integrated learning method algorithms based on decision tree, Deep Forest, LightGBM, and XGBoost was evaluated using Python language programming, and, finally, a four-dimensional vector early warning prediction model based on the Deep Forest algorithm for the total number of shrimp bacteria, the proportion of Vibrio shrimp, the total number of water bacteria, and salinity was established (accuracy: 89.00%). Although the prediction performance of the Deep Forest model decreased somewhat compared with that of the support vector machine model established in this study, the algorithm was gradually screened out based on the correlation between factors, including the effects of all factors. It was proven that the Deep Forest model established in this study was the ideal prediction model for predicting the occurrence of AHPND in P. vannamei among the 10 dimension parameters tried, and the superiority of the Deep Forest algorithm was also further verified. The results provide basic data and technical support for shrimp AHPND disease prediction, prevention and control, and lay a theoretical foundation for further establishment of aquaculture animal disease early warning theory.

Abstract:As a pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed widely in the marine environment, and the host species of PDD are diverse. Although in the past decade the number of reports on the pathogenicity of PDD to marine animals has gradually increased, it is necessary to study this further in order to establish targeted prevention and technical control measures to reduce harm to marine organisms. In this study, a high-virulence PDD strain (PDD1608) was selected to explore the effect of the virulence gene dly on the biological characteristics and pathogenicity. The dly-deleted mutant strain Δdly PDD1608::Cm was successfully constructed using the λRed recombination technique. The biological characteristics of the wild-type and mutant strains were compared, including growth, swarming motility, drug susceptibility, physiological and biochemical characteristics, biofilm formation ability, and hemolytic and phospholipase activity of extracellular products (ECP). Meanwhile, Oryzias melastigma was used as the target host, and the pathogenicity of wild-type and mutant strains and their ECPs to O. melastigma was detected by the artificial infection test. There was a positive correlation between the results of the bacterial solution and ECP challenge, indicating that deletion of the dly gene affects the virulence of the PDD strain. Deletion of the virulence gene dly resulted in slower growth of the PDD mutant strain; reduced swarming and hemolytic and phospholipase activity; no change in the drug susceptibility or physiological and biochemical characteristics of the wild type and mutant strains; compared with the wild type strain, significant different (P<0.05) biofilm formation ability of the mutant strain; the artificial infection test showed that the pathogenicity of the mutant strain and its ECP decreased. The results of this study revealed that the virulence gene dly affects many biological characteristics of PDD strains, such as growth, swarming motility, and hemolytic and phospholipase activity, and it was found to be closely related to the pathogenicity of the PDD strain and ECP. The results of this study contribute to further study of the influence of the virulence gene dly on the biological characteristics of the PDD strain in order to determine the appropriate prevention and control measures to curtail its spread and infection. The artificial infection test of the model organism O. melastigma helps provide an animal reference model for further exploration of the pathogenic mechanism and infection process of the PDD strain on the host. The results of the ECP experiments provide a theoretical reference for the research and development of PDD subunit vaccines.

Abstract:Flinders technology associates (FTA) card (Whatman®) is a paper-based matrix designed to fix, purify, and store genetic material from various biological sources. It can conveniently and quickly preserve nucleic acids and may fulfil the requirements of long-distance and cross-border sample transportation. The FTA card can store and transport tissue, nucleic acid, and other sample types at room temperature (20–25 ℃). Nucleic acid can be extracted directly for detection and be sent by express as an ordinary parcel without being treated as dangerous or as special goods, eliminating tedious processes, saving time, and ensuring sample quality. It is widely used in the human and animal medicine field. It has been successfully used for the storage and transportation of livestock pathogens and viral nucleic acids. In terms of aquatic animals, the FTA card has been used by researchers to store white spot syndrome virus (WSSV) and the shrimp Enterozoon hepatopoaei (EHP): However, there is no relevant research report on the elution effect of the nucleic acid stored in the FTA card, which affects the application of the FTA card. The infectious hypodermal and haematopoietic necrosis virus (IHHNV) is an important shrimp pathogen and severely impacts the shrimp culture industry. It was first found in Hawaii, United States, in 1981 and then spread to several countries, including to Australia, Singapore, Malaysia, South Korea, Brazil, and China. The IHHNV infecting Penaeus vannamei does not cause high mortality, but growth would become slow and deformed, resulting in great economic losses. Early detection and prevention management are particularly important in current situations, which lack effective control measures for the disease. Several IHHNV detection methods have been established that use molecular biological methods, including conventional polymerase chain reaction (PCR) and real-time PCR (these are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health). Nucleic acid extraction by the above methods meets the requirements for samples, usually frozen, ethanol, or other nucleic acid preservation reagents. Low temperature preservation conditions and composition restrictions of preservation solutions present certain difficulties in disease investigation, surveillance, and monitoring of shrimp farming. It is particularly important to address this dilemma. To find a fast method for the preservation and separation of IHHNV DNA and provide complete nucleic acid materials for subsequent research, we selected FTA cards as the preservation medium, and designed seven kinds of FTA cards with attached DNA elution methods based on the FTA purification reagent, TE buffer, and deionized water. We evaluated the elution and separation effects of different nucleic acids and the minimum amount of dot FTA card nucleic acids through real-time PCR detection. The appropriate solution was spotted onto FTA cards according to the manufacturer´s protocol, labeled, and air-dried for 1 day at room temperature. The result shows that on the 4 mm2 FTA card, the sample volume was 2.5 μL. When the eluent is used as the template, the minimum FTA card nucleic acid concentration needs to be 1.47×104 copies/μL above the best detection sensitivity, and 100% detection rate can be obtained by washing the FTA card with 50 μL TE buffer solution at 95 ℃ for 5 min. Using the FTA card as the template, the nucleic acid concentration of the dot FTA card needs to be above 1.82×103 copies/μL, eluted with FTA purified reagent thrice at room temperature, and then eluted with TE buffer twice. Each elution time is 5 min as this can obtain the best detection sensitivity and demonstrates 100% accuracy. The elution effect of the above two schemes was better than that of the other five schemes. The nucleic acids of WSSV, EHP, decapod iridescent virus 1, covert mobility Noda virus, and Vibrio parahaemolyticus causing acute hepatopancreatic necrosis were preserved using FTA card to test the efficiency of the established elution method. It is assumed that this method is universal for the elution of other shrimp pathogenic nucleic acids. At present, research on the application of FTA card is mostly seen in the nucleic acid effect of its preservation and transportation of tissue samples. There are few reports on the relationship between the amount of preserved nucleic acid, separation methods, and detection effect. This study shows that FTA cards used to preserve pathogenic nucleic acid requires a specific amount of nucleic acid in the sample and directly affects the detection results of the sample with different FTA card elution methods. This study provides a feasible scheme for the preservation and elution of IHHNV DNA with FTA cards. The application of this technology has potential use as storage and transport strategy for surveillance programs and can enhance biosecurity in shrimp culture, which provides a scientific basis for the preservation and transportation conditions for the collection of wild shrimp samples and the regional transmission of viral nucleic acid samples.

Abstract:The razor clam (Sinonovacula constricta, Class Bivalvia) is a kind of burial filter-feeding shellfish. Salinity fluctuation is an important source of pressure for water habitats. High salinity in some coastal areas of Shandong and Jiangsu impact the survival and germplasm conservation of razor clam. To study the ecological behavior response of S. constricta to high salt culture environment, two populations of razor clams were used, including "Shenzhe No.1" population (SZSC) and a natural population (ZRSC). The semi-lethal salinity level of each population was determined. The effects of control group (20) and high salinity (24, 28, 32) on burrowing and feeding behavior of razor clams were studied. The differences in burrowing indices and feeding physiology between the two populations were compared. In the burrowing behavior experiment, two groups were set; razor clams from the temporary pond were put into each salinity group to start the experiment, while the other group of razor clams were stressed under each salinity condition for 24 h and then put into each salinity group to start the experiment. The results showed that the 120 h LC50 of SZSC was 34.04, while the 120 h LC50 of ZRSC was 32.04. The burrowing behavior of razor clams could be divided into four periods: The preparation period of shell closure, the period of axe foot movement, mud digging period, and the end period of mud diving. In the non-stressed group, the burrowing time of 50% (BT50) of SZSC was significantly higher than that of ZRSC (P<0.05). The BT50 of SZSC at 24 salinity was the minimum, which was significantly lower than that of BT50 at 28 and 32 salinity. The distribution of burrowing depth of SZSC was highly concentrated: 50% of the individuals were between 7.29 and 7.55 cm. The burrowing rate was 88.33% at 32 salinity, which was significantly higher than that of ZRSC (P<0.05). In the stressed group, the BT50 of SZSC was significantly lower than that of ZRSC, while the burrowing rate was significantly higher than that of ZRSC (P<0.05). With the increase in salinity, the burrowing rate of ZRSC decreased significantly (P<0.05). The average burrowing depth of ZRSC was 7.45 cm at 32 salinity, which was significantly higher than that of SZSC (P<0.05). By comparing the experimental results of the two populations, whether in SZSC or ZRSC, the BT50 of razor clams in the stressed group was higher than that in non-stressed group at each salinity. However, there was no significant difference in BT50 between the stressed and non-stressed groups at 20 and 28 salinity among SZSC (P>0.05). The BT50 of SZSC was closer to that of the control group at 24 and 28 salinity, and the vitality of SZSC was significantly better than that of ZRSC after 24 h of salinity stress. Under high salinity, the distribution of SZSC in mud was more concentrated than that in ZRSC, and the burrowing depth was shallower. In terms of feeding physiology, the feeding rate of SZSC was significantly higher than that of ZRSC under high salinity (P<0.05). The feeding rate of SZSC reached the maximum 89.54 mL/(g·h) at 24 salinity, which was significantly higher than that of other salinity groups (P<0.05). In summary, the ecological behaviors of both populations were affected by high salinity. The higher the salinity, the stronger the stress response, with the SZSC having a higher salinity tolerance than ZRSC. In this study, the tolerance of two populations of razor clams to high salinity was evaluated at the level of ecological behavior, and the vertical distribution and feeding ability of S. constricta in sediment in a high-salt environment was revealed. The results provide a theoretical reference for the further breeding of novel high-salt-tolerant strains of S. constricta.

Abstract:Pen shell (Atrina pectinata) is a Bivalvia species with high commercial value in China. However, large scale production is difficult owing to the lack of seed. Pen shell uses external fertilization with sperm stored in the testis in a nonmotile state. Sperm motility is initiated when they are released from the reproductive tract into the aquatic environment, which enables fertilization. Various chemical signals including pH, ions, and cyclic nucleotides control sperm motility. However, there are interspecific differences in activation conditions between different Bivalvia species. Screening out an effective activating medium is the basis of artificial breeding technology. Swimming parameters, such as total motile sperm (TM), movement velocity, and beat-cross frequency (BCF) are common indicators to evaluate sperm quality. Sperm must have sufficient motility to reach the egg to complete fertilization. In addition, sperm needs a large amount of ATP to maintain its swimming movement. Intracellular ATP content controls the duration of the sperm movement phase in most marine species. Studying the movement characteristics and energy metabolism of sperm during activation will help develop and optimize artificial breeding technology. Studies of A. pectinata mainly focus on their oocytes. However, the activation conditions, moving characteristics, and energy metabolism of sperm during activation remains unknown. Thus, there is an urgent need to screen appropriate media and study the activation mechanisms of A. pectinata sperm. Adult A. pectinata were collected from Wuzhizhou Island, Hainan Province in November 2021. Artificial seawater with different levels of ammonia ions and pH were used to activate sperm. This study examined A. pectinata sperm activation in artificial seawater by varying the ammonia ion concentration and pH. The change in sperm motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), and BCF were described. The ATP content, ATPase activities, and superoxide dismutase (SOD) activity were quantitatively recorded during the full activated stage. The motility was slightly improved by increasing the pH of seawater, but could not achieve the fully activated stage. Furthermore, the motility was significantly improved when activated by alkalized seawater containing ammonia ions, and the best results were observed in groups containing 3 mmol/L ammonia ion: The sperm motility index (MI) was in the fully activated stage (MI ≥ 4) until the end of the experiment, with TM ≥ 80%, VCL > 56 μm/s, VSL > 17 μm/s, VAP > 30 μm/s, and BCF > 6 Hz. Sperm ATP content decreased to 30.29% of their initial values [(128.80±66.92) μmol/g prot] during 5 min post activation and was maintained at this level during post-activation. ATPase activities were maintained at a constant level. Na+-K+-ATPase activity was lower [(0.62±0.03) U/mg prot] compared to Ca2+-Mg2+-ATPase activity [(6.08±0.04) U/mg prot]. The SOD activity of sperm steadily decreased to [(1.23±0.73) U/mg prot] during 15 min post activation and remained stable. In conclusion, pH was not the decisive factor in pen shell sperm motility. Instead, ammonium ion promoted sperm activation. There was a significant decrease in sperm ATP concentration (P<0.05) at the beginning of the post-activation stage and the ATP concentration followed by stabilization at a lower level. The reduction in SOD activity may cause oxidative stress. The findings in this study can be instructive to conduct further research on sperm activation mechanisms, and help develop artificial breeding technology for A. pectinata.

Abstract:Aquaculture is the fastest growing food production industry. The Chinese mariculture industry has made great progress in the past 40 years to become the dominant producer of aquaculture. However, the current deterioration of the water environment and the aggravation of farming diseases poses challenges for the traditional culture model to meet the new requirements of healthy development in marine fishery resources. This problem was addressed using marine ranching. This is a new mariculture model that includes two approaches (artificial reefs and stocking) with the goal of achieving environmental and ecological harmony. Artificial reefs are an important component of this marine ranching model since they can help improve the water environment around the reefs, promote nutrient circulation, provide a suitable habitat for marine organisms, and encourage colonization of the reef surface by sessile organisms. The placement of artificial reefs into seawater may serve as a substrate for bacteria to form a biofilm on the surface. Biofilms play a crucial role in the settlement of many marine invertebrate species. However, limited research was conducted into the relationship between bacteria on the surface of artificial reefs, biofilm formation, and the settlement behavior of Mytilus coruscus. This study placed white acrylic plates and tetrahedral structured artificial reefs in Gouqi Island, Zhoushan City, Zhejiang Province, China (122°46′ E; 30°43′ N) . Nine strains of bacteria isolated from the surface of the artificial reefs were used to construct mono-species bacterial biofilms and induce plantigrade settlement of M. coruscus. The aim of the study was to investigate the interactions between marine bacteria and the settlement of M. coruscus on the surface of artificial reefs. The marine biofilms impacted the settlement process of M. coruscus. The bacterial species from the marine biofilm were screened for high and low inducing activity and analyzed for bacterial density, protein, and polysaccharide content to further explore the relationship between different bacterial biofilms and M. coruscus settlement. There were significant differences in the induction activity of biofilms formed by the nine strains of artificial reef bacteria on the settlement of M. coruscus: Mesoflavibacter sp.2 and Phaeobacter sp.2 showed the highest and lowest induction activity, respectively. Bacteria within the same genus exhibited differences in induction activity. This indicated that the induction activity of bacterial biofilms on mussel settlement was independent of bacterial species. Phylogenetic analysis showed that genetically similar strains (such as Pseudoalteromonas sp.31 and Pseudoalteromonas sp.32) and genetically distant strains (such as Mesoflavibacter sp.2 and Jeotgalibacillus sp.1) showed significant differences in the induction activity of M. coruscus settlement. Only four of the nine bacterial strains showed significant correlation between biofilm density and the settlement rate. Sutcliffiella sp.1 and Jeotgalibacillus sp.1 exhibited a positive correlation between bacterial density and induction activity. This indicated that bacterial density may play a role in M. coruscus settlement, although it may be strain specific. Additionally, the trend of induction activity of mussel larvae attachment varied with increasing initial bacterial density. The optimal density for settlement is strain specific. Further analysis of biofilm active substance content in Mesoflavibacter sp.2 and Phaeobacter sp.2 revealed that polysaccharide content negatively correlated with induction activity of M. coruscus and positively correlated with protein content. This suggests that bacterial species may not directly affect M. coruscus settlement, although bacteria may indirectly influence settlement by affecting the secretion of extracellular products. This study showed that nine bacterial strains had significant differences in their ability to induce mussel settlement on the surface of artificial reefs. Interestingly, these differences did not necessarily correlate with the genetic distance between the marine bacteria. Further investigations were conducted on two selected strains of bacteria (Mesoflavibacter sp.2 and Phaeobacter sp.) that exhibited different levels of inductive activity on M. coruscus. The polysaccharide content and protein content negatively and positively correlated with the induction activity of M. coruscus, respectively. This suggests that the presence of specific polysaccharides may negatively affect the settlement of M. coruscus according to cellulose content measurements. This is the first study investigating the effect of bacteria on M. coruscus settlement on the surface of artificial reefs. This has significant theoretical implications for further research on the interactions between biofilms and marine invertebrates on the surface of artificial reefs in natural marine environments. Understanding the settlement mechanisms of marine benthic organisms on artificial reefs is crucial to manage and conserve marine resources since artificial reefs are widely used to enhance marine habitat and biodiversity. The findings of this study have practical implications for the design and construction of artificial reefs. Understanding the role of bacteria in mussel settlement facilitates the optimization of artificial reef structures to promote or inhibit the settlement of target species. This knowledge can help develop effective strategies to manage biofouling on artificial reefs. This can impact the performance and longevity of these structures. Further research in this field will deepen our understanding of the underlying mechanisms and enable development of management strategies for artificial reefs and marine conservation efforts.

Abstract:Ultraviolet mutagenesis is a safe and efficient method to induce mutations in laver. It has the advantages of non-pollution, high efficiency, easy operation, and low cost. It has been primarily used for mutagenesis of the filament, protoplast, or blade of cultivated Pyropia species, but is rarely used to induce genetic mutants from germinating conchospore. The germination period of conchospores is the period of meiosis in Pyropia haitanensis. The four progeny cells of germinating conchospores are linearly arranged forming a meiotic tetrad. The tetrad cells that undergo genetic recombination can determine the developmental pattern and segregation of traits in the thallus. In this study, short-wave ultraviolet (UV-C) irradiation with different doses (50, 100, 200, 300, 400, 500, and 600 J/m2) was used to induce color mutants during the germination of conchospores in P. haitanensis. The results showed that low-dose irradiation (50 J/m2) promoted the germination of conchospores, while irradiation doses above 100 J/m2 inhibited the germination and growth of tetrad germlings. Therefore, low-doses of UV-C irradiation were used in the production to promote the germination of conchospores and improve the production efficiency of laver. In the dose range of 50–400 J/m2, the frequency of color chimeras increased with increasing irradiation intensity. When the dose was 300 J/m2 and 400 J/m2, and the pigmentation mutation rate was 15.22% and 17.18%, respectively, and the death rate of conchospores was 51.70% and 61.00%, respectively. In the dose range of 50–500 J/m2, with the increase in UV-C irradiation dose, the proportion of 2-color sectored chimera showed a trend of first decreasing and then increasing, and the proportion of 3- and 4-color sectored chimera showed a trend of first increasing and then decreasing. Among them, the regenerated color chimeras that appear were generally 2- and 3-color sectored chimera, yet the proportion of 4-color sectored chimera was the least. When the irradiation intensity reached 500 J/m2, the majority of the conchospores died, and the death rate was 83.98%, and the frequency of color mutants was significantly lower than that of 300 J/m2 and 400 J/m2 dose groups. These results indicate that the mutagenesis effect was the best when the dose was 300 or 400 J/m2, which was convenient to obtain abundant genetic recombination and genetic variation in progeny cells. In addition, UV-C irradiation also had a significant effect on the early development of conchospores and phenotypes of pigmentation mutant arranged in tetrad germlings, which was mainly manifested in the large number of color-sectored blades developed from the irradiated conchospores. Simultaneously, UV-C irradiation retarded the development of cells at the top of conchospore germlings, inhibited the development of cells at the middle base toward the poles, and increased the lateral development, resulting in the decrease in blade aspect ratios. Somatic cell germlings with single colored pigmentation were also isolated from color sectored chimeras by enzymatic hydrolysis. The color species of the five monochromatic mutants obtained were basically consistent with the pigment variant sectors observed on color-sectored thallus, indicating that the obtained color mutants were derived from a single mutant cell on the maternal color-sectored thallus. In conclusion, UV-C irradiations can effectively mutate the conchospores of P. haitanensis, and appropriate irradiation doses (300 or 400 J/m2) can obtain a certain number of color-sectored thallus. This study provides a novel pathway for the preparation of artificial color mutants and mutation breeding in P. haitanensis.

Abstract:In the Healthy China Strategy context, fish are increasingly in demand as a source of high-quality protein. Silver carp (Hypophthalmichthys molitrix) is a resource-rich and highly productive freshwater fish species found in China that is not edible raw or cooked in its original form due to its many boney spines. However, due to its advantages of being low-cost, low in fat, and high in protein, it is an ideal choice for surimi production. Currently, it is widely used in the industrial production of surimi products. The development of the freshwater surimi industry can significantly improve the added value of freshwater fish utilization, which has attracted extensive attention. Freshwater surimi is high in protein and low in fat and has a smooth and delicate taste, making it extremely popular with consumers. It has a high output, low price, is growing in demand, and is gradually being accepted throughout domestic and foreign markets. It has also driven the development of some related industries and produced significant economic and social benefits. As domestic consumers experience improved living standards and a faster pace of work, premade dishes containing surimi products as well as recreational snack surimi products with increased shelf-life are more attractive, as they save the consumer processing time, are enjoyed by the consumer, and meet their nutritional demands, affording these products great market potential. With the development of surimi products and related industries, specific requirements are being put forward for its production. Although China supplanted Japan as the largest producer of surimi products worldwide in 2006, ushering in a period of nearly 10 years of high production growth, the annual production of surimi products in China since 2014 has stagnated or even slightly decreased. Moreover, the surimi industry has entered a bottleneck period for quality enhancement caused by the expansion of quantity. The fishy odor of freshwater surimi is one of the industrial problems that affect the quality and efficiency of surimi. The flavor of surimi products (such as fish balls, fish intestines, fish cakes, and others in hot pot) has become one of the quality attributes that consumers are extremely concerned about. However, the adsorption and release laws of key odor-active substances are still unclear. There are existing research technologies for surimi odor, mainly including instrumental analysis (gas chromatography-mass spectrum, gas chromatography-olfactometry, gas chromatography-olfactometry- mass spectrum, electronic nose technology, etc.), sensomics analysis (odor activity value (OAV), aroma extract dilution analysis, odor recombination, odor omission test, etc.), and enzyme-linked immunosorbent assay. The research objects of odor sensory experiments are mostly rice wine, oil, vegetables, fruits, and fungi. Moreover, present odor research is mainly carried out in a prepared solution, mainly using odor recombination of a liquid simulation system, which is different from the interaction between the real odor active substance-solid surimi. Therefore, constructing an odor model based on solid surimi is necessary to better simulate the sensory characteristics of surimi. To build an odor model based on solid surimi, an odorless or low-odor surimi background model must be established in order to investigate the interaction between various odor components and surimi. There are several fishy substances and complex components in freshwater fish and surimi products, including aldehydes, alcohols, ketones, esters, sulfur compounds, nitrogen compounds, and alkanes. At present, most studies on rinsing surimi reported worldwide are based on how to better apply it to the food system, ignoring interactions between components in the complex system of surimi, which creates certain limitations in establishing a background model of surimi. Therefore, salt, salt-alcohol, acid, alkali, and other rinsing media were selected in this study, which was not limited to food systems. By comparing the removal effects of different rinsing media on the odor residue of surimi, an odorless or low-odor background model of surimi could be constructed. Here, the effect of different rinsing media on the odor residue of a surimi background model was studied. Specific rinsing media were as follows: 0.5% NaCl (W/W) + 0.35% Na2CO3 (W/W) + 4.0% C2H5OH (W/W) solution (group A), 0.5% NaCl (W/W) + 0.35% Na2CO3 solution (group B), 0.5% CaCl2 (W/W) + 0.35% Na2CO3 (W/W) + 4.0% C2H5OH (V/W) solution (group C), 0.5% CaCl2 (W/W) + 0.35% Na2CO3 (W/W) solution (group D), 1% NaCl (W/W) + 1% Na2CO3 (W/W) + 4.0% C2H5OH (V/W) solution (group E), 1% NaCl (W/W) + 1% Na2CO3 (W/W) solution (group F), 1 mol/L HCl solution (group G), and 1 mol/L NaOH solution (group H), respectively. The results showed that SPME-GC-MS detected 65 volatile compounds in silver carp surimi, including 22 aldehydes, 13 alcohols, 9 ketones, and 7 hydrocarbons, among which the contents of aldehydes and alcohols were high, which had a major contribution to the odor of silver carp surimi. A total of 18 odor-active substances were detected by the OAV (≥1) method, which helped illustrate that odor-active compounds contribute to the overall odor of surimi. After treatment with eight kinds of rinsing media, the residual amount of the odor-active substances in the silver carp surimi was washed or released to varying degrees, affecting the sample's overall odor contribution. The rinsed surimi samples contained 6, 8, 7, 9, 6, 12, 9, and 9 odor active compounds and residual rates of volatile odor compounds were (0.380±0.120)%, (0.610±0.086)%, (0.280±0.033)%, (0.480±0.037)%, (0.150±0.018)%, (4.330±0.160)%, (18.680±0.081)%, and (0.490±0.003)%, respectively. According to the SPME-GC-MS analysis results, due to the synergistic effect of ethanol, the content of volatile compounds detected in group E was the lowest, the total residual amount of odor-active compounds was reduced to (6.57±0.77) μg/kg, and the total residue rate was only 0.15%. Meanwhile, the total OAV decreased to 2.52±0.25, there were 17 odor-active substances with an OAV<1, and the OAV of nonanal was only 1.34±0.05, which could establish a low-odor background model of surimi. Furthermore, electronic nose and sensory evaluations distinguished the overall odor characteristics between different rinsed samples and fresh surimi. This study took silver carp surimi as the research object and studied the influence of volatile odor compounds and salts, salt-alcohols, acids, alkalis, and other rinsing media in surimi on the residual rate of odor substances through SPME-GC-MS, electronic nose, and sensory evaluation methods, which will significantly contribute to the establishment of an odorless or low-odor solid surimi model and provide a novel idea for sensory analysis.

Abstract:Yellowfin tuna (Thunnus albacares) is popular with consumers due to its delicious meat and high nutritional value. It is a globally recognized high-end marine economic fish. With the advancement of fishing equipment and the increase in fishing efforts, the amount of tuna resources has decreased significantly. To make up for the insufficient supply of tuna in the market and protect wild populations, it is essential to carry out research on artificial aquaculture technology for tuna and to gradually establish full-cycle cultures of tuna. In order to study the differences in the nutritional components and quality of yellowfin tuna muscles of different sizes, three different-sized yellowfin tuna, J1 (4.2±1.2) kg, J2 (22.5± 2.5) kg, and J3 (50.8±3.9) kg, were used as the research subjects, and conventional biochemical analysis methods were used to compare and analyze the proximate compositions, amino acids, fatty acids, and mineral elements of tuna muscle. The results showed that (1) the moisture level of the J1 group was significantly higher than that of the J2 and J3 groups (P<0.05); the crude protein levels of the J2 and J3 groups were significantly higher than that of the J1 group (P<0.05); and the crude fat level in the J3 group was significantly higher than that in the J1 and J2 groups (P<0.05). (2) A total of 19 common amino acids were detected in yellowfin tuna muscle, including nine essential amino acids (EAA), two semi-essential amino acids, and eight nonessential amino acids. The amino acid with the highest content was glutamic acid (3.04~3.25 g/100 g) of the three tuna sizes; the essential amino acid with the highest content was lysine (2.02~2.15 g/100 g), and the essential amino acid with the lowest content was tryptophan (0.31~0.45 g/100 g). The amino acid content of different-sized yellowfin tuna varied greatly, except for threonine, valine, methionine, isoleucine, tyrosine, and phenylalanine, which were not significantly different among the groups, and the other amino acid contents were mainly J3>J2>J1. The content of nonessential amino acids was J3>J2>J1 (P<0.05). The content of essential amino acids and delicious amino acids in the J1 group was significantly lower than that in the J3 group (P<0.05). The content of semi-essential amino acids in the J1 group was significantly lower than that in the J2 and J3 groups (P<0.05). The ratio of EAA/TAA (total amino acids) in each group was above 40%, and there were no significant differences. According to the Amino acid score (AAS) score, the valine score in each group was the lowest and < 1, which was the first limiting amino acid. According to the chemical score (CS) score, except for lysine and tryptophan of the J3 group, the scores of other amino acids were all less than 1. The first limiting amino acid of groups J1 and J2 was tryptophan, while the first limiting amino acids of the J3 group were phenylalanine + tyrosine. (3) A total of 25 fatty acids were detected in the muscles of yellowfin tuna of three sizes, including 10 saturated fatty acids (SFAs), 5 monounsaturated fatty acids (MUFAs), and 10 polyunsaturated fatty acids (PUFAs). There were nine fatty acids whose content was greater than 1%, and their average content ranged from high to low: C22:6n3 docosahexaenoic acid (DHA), C16:0, C18:0, C18:1n9c, C20:5n3 eicosapentaenoic acid (EPA), C20:4n6 arachidonic acid (ARA), C18:2n6c, C24:1n9, and C16:1n7. Among these nine fatty acids, only C16:0, C24:1n9, and C16:1n7 showed no significant differences (P>0.05). The fatty acids in yellowfin tuna muscle were mainly PUFAs, and the content of DHA accounted for 37.46%~39.18% of the total fatty acid content. The DHA content of the J3 group was significantly higher than that of the J1 and J2 groups (P<0.05). The EPA content of the J2 and J3 groups was significantly higher than that of the J1 group (P<0.05). The DHA:EPA ratio of the J1 group was significantly higher than that of the J2 and J3 groups (P<0.05), and the MUFA content was J3>J2>J1 (P<0.05). The PUFA content of the J3 group was significantly higher than that of the J1 and J2 groups (P<0.05). PUFA/SFA and n-3/n-6 ratios of the J2 and J3 groups were significantly higher than those of the J1 group (P<0.05). The h/H ratio of the J3 group was significantly higher than that of the J1 and J2 groups (P<0.05). (4) Among the four macro elements, the contents of sodium and calcium of the J2 and J3 groups were significantly higher than those of the J1 group (P<0.05), and the potassium content of the J1 group was the highest and was significantly higher than that of the J2 and J3 groups (P<0.05). The contents of four heavy metal elements were all far lower than the maximum allowable limit (FAO and WHO) suggested in food, among which the iron content was the highest in the J3 group, and J3>J2>J1 (P<0.05). The copper content was the highest in the J3 group, and was significantly higher than that in the J1 group (P<0.05). Comprehensive analysis showed that large size yellowfin tuna had better nutritional quality, and the results of this study also provide a scientific basis for the selection of residents' diets and the formulation of artificial nutrition feed for yellowfin tuna.

Abstract:Flavor is an important characteristic of seafood products, and drying can produce unique pleasant flavors. Drying is among the most common methods for processing seafood products. It can improve quality and shelf-life of seafood products and produce unique flavors. Oxidative hydrolysis of lipids during dry fish processing in the presence of light, photosensitizers, heat, oxygen, transition metal ions, and microorganisms produces volatile small molecules, including alcohols, ketones, aldehydes, and acids, which contribute to the flavor profile of dried fish. Volatile compounds are important components of seafood flavor. Flavor analyses are usually performed using gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography-mass spectrometry (GC-MS) in combination with electronic nose/tongue techniques, which not only characterizes the molecular composition of volatile components in the sample, but also yields macroscopic results via the electronic nose/tongue, ultimately combining instrumental analysis with quantitative sensory data for a comprehensive evaluation of sample flavor. Currently, the market sales model of Cololabis saira is mainly based on a single frozen whole C. saira, and excludes most types of deep-processed products. There is an urgent need to enrich research into processing effects on C. saira quality and flavor, and further develop markets for deep-processed C. saira products. To explore the effects of different drying methods on C. saira flavor, we assessed flavor molecule profiles using GC-MS and electronic tongue techniques. This study aimed to provide a theoretical basis for improving C. saira product flavor, thereby enhancing the economic impact of the C. saira industry. In this study, C. saira was thawed in low-temperature air, and the giblets were removed and diagonally cut. Pre-treated fish were then soaked in 15% salt water for 1 h, drained naturally, and subjected to natural drying (natural air-drying on a sunny day in autumn for 3 days, environmental temperature 10~20 ℃, humidity 25%~42%), cold air-drying (continuous cold air-drying for 3 days, setting temperature (15±2) ℃, relative humidity 38%~40%), and UV with cold air-drying (continuous UV with cold air-drying for 3 days, ultraviolet lamp irradiation, setting temperature (15±2) ℃, relative humidity 38%~40%). The flavor profiles of fresh fish (CK), cured fish (0 d), naturally dried fish (N), cold air-dried fish (C), and UV treated cold air-dried fish (U) were compared. Significant differences were observed in the odor and taste of dried C. saira among products of the different drying methods. GC-MS results showed that a total of 58 volatile flavor substances were detected, including aldehydes, alcohols, ketones, acids, hydrocarbons, and nitrogenous compounds. Increased alcohols, aldehydes, and ketones enriched the fatty aroma of the three dried C. saira samples to varying degrees. Among them, the contents of cis-2-heptenal, octylaldehyde, 2-ethylfuran and other substances in U group increased significantly, increasing to 64.96, 569.48 and 189.27 μg/kg, respectively, so that the U group had richer fat flavor. Hexanal, heptanal, Z-4-heptenal, octanal, nonanal, (E,E)-2,4-heptadienal, (E,E)-2,6-nonandialdehyde, 1-octen-3-ol, heptanol, 2,3-pentanedione, 3,5-octadien-2-one, and trimethylamine were the odor-active substances common to the five C. saira samples and were used as flavor compounds to characterize the oily and fishy taste of C. saira. E-2-nonenal, 2-ethylfuran, E-2-octenal, 2-nonanone, 2-undecanone, and 1-nonanol are three odor-active substances specific to dried C. saira, with E-2-octenal, 1-nonanol and 2-undecanone, which have an oily smell, and 2-ethylfuran, which has a burnt smell, having the highest odor aroma-active in the U group. Salty taste, richness, bitterness, astringency, and sourness of the fish increased after the drying process, especially salty taste and richness. Only fresh taste was significantly reduced relative to fresh fish. Saltiness, freshness, and richness of dried fish are important taste indicators. Salty taste and richness increased significantly after the three drying processes, whereas freshness decreased. Group U exhibited the highest salty taste and richness. In conclusion, the volatile odor and profile of C. saira changed significantly with each of three drying processes (natural drying, cold air drying, and UV with cold air drying), all of which increased the fatty flavor and considerably reduced the fishy flavor. Moderate oxidation positively contributes to C. saira flavor. Increased fatty flavor reduces the proportion of fishy substances, thus improving C. saira flavor. UV irradiation with cold air drying promoted lipid oxidation to some extent, producing more fatty substances, as well as cis-2-heptenal and 2-ethylfuran, which enriched the roasted, charred flavor of dried C. saira. Salinity, freshness, and richness are important taste indicators of dried C. saira. All three drying methods enhanced the salinity and richness of C. saira, and UV irradiation with cold air-drying significantly improved the salinity and richness of the fish and enriched its taste and aftertaste. Therefore, among the three drying methods, the method involving UV with cold air drying significantly enriched the flavor of C. saira to the greatest extent.