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Abstract:Due to climate change, the biomass and biodiversity of fishery resources are increasing at higher latitudes and decreasing at lower latitudes (reflected by the mean temperature of the biomass, MTB). Studies have shown that the change in catch composition of marine fisheries at the global scale is largely related to seawater temperature. It remains important to accurately analyze biodiversity and biomass distribution to inform the sustainable utilization and adaptive management of fishery resources. To evaluate the long-term spatiotemporal dynamics of biodiversity and MTB of fishery resources in the Yellow Sea, we selected species that account for more than 0.05% of the total biomass as representative species based on scientific bottom trawling data in autumn (October) of 2000, 2009, and 2018. A total of 117 stations (39 stations each year) were surveyed by R/V Beidou. Considering that sea surface temperature (SST) is the most accessible oceanographic variable and has been shown to affect marine biomass, the impact of SST on the biodiversity of benthic fisheries in the Yellow Sea was analyzed. Therefore, the spatiotemporal distribution of fish, crustaceans, and cephalopods, their relationship with SST, and the spatiotemporal distribution of biomass and MTB were analyzed. The Shannon-Wiener, Berger-Parker, and Margalef biodiversity indices were used to evaluate distribution changes. Location-related data were matched at the same resolution (0.5°×0.5°) for further analysis. Data processing and plotting were performed using R. The results showed that 39 species in 2000, 37 species in 2009, and 46 species in 2018 were representative of the total biomass collected by bottom trawling. The increase in diversity observed in 2018 may have resulted from a decrease in the abundance of dominant species. Although Liparis tanakae was dominant between years, there were significant differences in biomass proportions: 27.00% in 2000, 37.85% in 2009, and 22.82% in 2018. Fish showed the highest richness around 33°~34°N, and that of the southern Yellow Sea was higher than the northern Yellow Sea; crustaceans showed higher richness south of 34°N, and gradually increased from 2000 to 2018. For cephalopods, Japanese flying squid (Todarodes pacificus) was the only species occupying more than 0.05% of the total biomass. Except for cephalopods, the richness of all categories increased northward. According to the species accumulation curve, approximately 20 random stations were needed to represent Yellow Sea species richness. The Rényi profile further verified that 1) species were not evenly distributed, 2) species richness decreased from 2000 to 2009 and increased in 2018, and 3) the proportion of the most dominant species was highest in 2009 and lowest in 2000. In terms of biomass, high biomass stations occupied only one-third of the total stations and were mainly distributed around 35°~37°N in 2000, while high biomass stations occupied half of the total stations in 2009 and 2018. The proportion of fish was >70% in all years with a downward trend, followed by an upward trend; the proportion of crustaceans increased from 11.45% to more than 25%; the proportion of cephalopods was the lowest (less than 1%) with a downward trend. Previous studies have also shown that crustaceans gradually dominate over time in both abundance and biomass. In particular, the abundance of Crangon affinis was dominant in all years, accounting for 53.25% in 2000, 75.40% in 2009, and 63.81% in 2018. In terms of the spatiotemporal distribution of biodiversity indices, the Berger-Parker index and Shannon-Wiener index showed contradictory distribution trends; high Shannon-Wiener and Margalef index values were mainly distributed in the southern Yellow Sea, whereas high Berger-Parker index values were mainly distributed in the middle and southern Yellow Sea. For the same diversity index, Berger-Parker had the lowest values in 2018, while the Shannon-Wiener index and Margalef index values increased over time, and no obvious longitudinal or latitudinal change was found for any of the indices. There was no significant linear relationship between the SST and biodiversity indices (P>0.05). SST had a weak positive correlation with species richness, the Margalef index, and the Shannon-Weiner index, while SST had a weak negative correlation with the Berger-Parker index. Therefore, we concluded that bottom species are not sensitive to changes in SST, and bottom sea temperature should be compared to diversity indices in future studies. In terms of MTB, it was higher in the southwest and lower in the northeast with an obvious change around 34°N; it was lower in the deep-water area than in the coastal area; the lowest value appeared in the northern survey area, while the highest value appeared in the southern survey area. Considering the weak relationship between SST and diversity indices, SST is not feasible for diversity studies of bottom species. Research on the relationship between bottom sea temperature and diversity and the spatiotemporal distribution of bottom temperature-based MTB are needed in the future.

Abstract:The Yellow River estuary is one of the three largest estuaries along the coastal waters of China, along with the Yangtze and Pearl River estuaries. Its adjacent seawaters are rich in biological resources, with abundant fish habitat, spawning grounds, and migration channels in the Bohai Sea and the Yellow Sea. However, as the waters are too shallow for a survey ship, data was insufficient for research in the intertidal zone of the salt and freshwater interchange. Instead, by interviewing and analyzing the daily catches by the set net of the local fishermen, this study explored the fishery biodiversity in the intertidal zone of the Yellow River estuary from April to November 2020. Yellow River estuary is rich in fishery resources, especially small fishery organisms and fish recruitment stocks. There were monthly variations in the dominant species composition and biodiversity index. A total of 61 species were recorded, most of which were fish and crustaceans. The diversity, evenness, and species richness index increased in summer (July and August) and decreased in autumn (October and November). By cluster analysis of the species compositions, the surveyed months can be divided into two groups: May to September, and October to November. Lateolabrax maculatus and Liza haematocheila were the most common and dominant species in all surveyed months. The main contributors to the catch of each month were: snails from April to July, snails and fish in August, fish in September, and crabs from October to November. Catches by the set net were mainly composed of individuals with body mass <2 g from April to September. From body mass-frequency distributions, fish with body mass <10 g were the main contributors to total catches from April to September. The mean body mass of fish in all surveyed months was 13 g/ind.. The dominant fish species with body mass with <20 g were: Gobioidei in April; L. maculatus in May; Konosirus punctatus in June; Pennahia argentata, Gobioidei, and L. haematocheila in June to August; and Sardinella zunasi, Thrissa kammalensis, T. mystax, Sillago sihama, Tylosurus melanotus, Gobioidei, and L. haematocheila in September. From the above results, the local set net fishery survey proved to be an effective method of collecting fishery data in the intertidal zone and can be incorporated in studies considering the distribution of fishery resources and breeding grounds in the seawaters adjacent to estuaries. In addition, this study provided clear evidence that set net fishing practices could severely impact the natural recruitment and stock enhancement of fish resources due to the low selectivity of nets and the overlap with larval fish migration channels. The number of larval fish captured by the set net fishery was, roughly estimated, several times higher than the local enhancement released. To minimize the effects of net mesh size, considering the spatial-temporal distributions of fish larvae, set net fishing practices should be concentrated to summer and early autumn, though further research is required to comprehensively account for both the protection of fishery resources and livelihoods.

Abstract:In recent years, fishery species have decreased in size and quality in the Yellow River estuary seas due to the effects of climate change and human activities, such as water and sediment regulation of the Yellow River, overfishing, and sewage discharge, among others. At the same time, the fishery resource structure has changed greatly: Small-sized fish with low value, shrimps and crabs became the main catches. To conserve natural resources and maintain local fishery harvests, conservation efforts such as stock enhancement and fishing quotas have been employed. Research on the biological characteristics of important fish can provide a scientific basis for the rational utilization and stock enhancement of fishery resources in the future, and it can also offer support for making relevant fishery policies. Liza haematocheila is an economically important fishery species in the Yellow River estuary and mainly feeds on zooplankton, phytoplankton, and organic detritus, which limits eutrophication and promotes energy and carbon cycling at the ecosystem level. The species plays an important role in ecosystem function and the maintenance of biodiversity. This study investigated the biological characteristics of L. haematocheila to provide a scientific basis for its conservation and rational utilization in the Yellow River estuary. Furthermore, it can provide basic biological data for the study of ecological restoration in the Yellow River estuary. This study evaluated the distribution of body length and weight, the relationship between body length and weight, reproduction, growth, death, and development. All of the analyses were based on sampling data from set nets between April and November 2020. The body length range of L. haematocheila was 45~460 mm, the average body length was 149 mm, and the modal body length range was 55~185 mm. The body weight range was 2~1100 g, the average body weight was 83 g, and the modal body weight range was 2~80 g. Both the average body length and body weight were the highest in May and the lowest in September. Overall, small individuals were the most common, and individuals with a body length of less than 200 mm accounted for more than 78%. Analysis of variance showed significant monthly differences in both average body length and weight of L. haematocheila. The relationship between body length and body weight showed a strong correlation within and between months [W=3.17×10‒5L2.82, (R2=0.980 5, n=984)]. The growth of L. haematocheila was negatively allometric, with the growth of body length being faster than that of body weight. The body condition of L. haematocheila decreased with time, though it increased in July and fluctuated with a downward trend from July to November. The nadir body condition was observed in June (1.102±0.173) and the peak body condition was observed in July (1.535±0.467). The rate of gonadal development varied from month to month, and the proportion of individuals who did not reach gonadal maturity was large, with an undeveloped gonad proportion of more than 50% per month. There was a peak spawning period from April to May. The growth curve of L. haematocheila was fitted, and its growth parameters were estimated using the ELEFAN method. The total death coefficient of the population was estimated using a length-converted catch curve, the natural death coefficient was estimated using the empirical formula of Pauly, and the population development rate was calculated. The results indicated that the asymptotic length (L∞) of L. haematocheila was 608 mm, the growth rate (K) was 0.31 a−1, total death coefficient (Z) was 1.42 a−1, natural death coefficient (M) was 0.51 a−1, fishing death coefficient (F) was 0.91 a−1, and its population development rate (E) was 0.64. The L. haematocheila resource was overfished in the shallow coastal waters of the Yellow River estuary, and its open fishing length (L50) was only 57.4 mm in this production survey. Fixed-net harvesting led to overfishing in the Yellow River estuary from April to November 2020, with juvenile fish and supplementary stocks experiencing the greatest impact. We should reconsider the management strategies for L. haematocheila in the Yellow River estuary, and promote stock enhancement at the same time. Artificial propagation technology for L. haematocheila should be considered for supplementing L. haematocheila in the Yellow River estuary seas. Research on the release time, release of krill size, population characteristics, growth, and development should be addressed to provide an informed and scientific basis for stock enhancement.

Abstract:Aquaculture in China is spread over a total area of 7.108 5×106 hm². Nowadays, aquaculture is done with a focus on high bait utilization efficiency. Moreover, the proportion of high-density farming organisms and high investment in aquaculture is increasing every year. However, the negative environmental problems associated with high efficiency of aquaculture are often ignored, especially the eutrophication caused by nitrogen and phosphorus pollution. An aquaculture system is a reservoir of nitrogen and phosphorus and sediments play an important role in regulating nitrogen and phosphorus levels in the system. The occurrence forms of nitrogen and phosphorus are closely related to the adsorption and release rates of nitrogen and phosphorus. Benthic animals contribute to the organic composition of sediments, and biological interference is an important factor affecting the release of nitrogen and phosphorus. Perinereis aibuhitensis Grube is a typical benthic animal living in sediments, which feeds on humus and benthic algae, and is one of the main types of organisms that is artificially bred in China. P. aibuhitensis can change the physical structure of sediments by burrowing and through bioturbation; it can also transport dissolved oxygen from the upper water level to the depths of sediments and increase the redox potential of sediments. Studies of Meng et al (2020) have shown that different specifications of P. aibuhitensis have significant differences in the distance covered by the sediments. Significant differences are also present in pump water, pump water rate, axial crawling speed, and nutrient salt dissolution efficiency. The optimum stocking density of P. aibuhitensis is 330–500 g/m². Investigation of the occurrence pattern and vertical distribution of nitrogen and phosphorus in sediments is essential for assessing the endogenous nitrogen and phosphorus load in sediments, controlling eutrophication of water bodies, and assessing the risk of nitrogen and phosphorus nutrient release in sediments. In this study, the effects and role of P. aibuhitensis on the transformation of nitrogen and phosphorus speciation in sediments were measured, and the feasibility of its application as a bioremediation species in pollution control was evaluated. This study can provide theoretical basis and technical support for green aquaculture and environmental governance. In order to study the effect of biological disturbance of P. aibuhitensis on the occurrence pattern of nitrogen and phosphorus in sediments, experiments involving P. aibuhitensis with different characteristics were performed. The experiments were divided into four groups with 400 g/m2 biomass of P. aibuhitensis: A small size group [(0.7±0.3) g]; a medium size group [(2.5±0.3) g]; and a large size group [(4.3±0.3) g] as the treatment group; and a no-sand silkworm as the control group. The experimental period was 45 days. Sediment samples were collected in PVC tubes (inner diameter: 1.5 cm), and the collected samples were divided into the upper layer, middle layer, and lower layer by partitioning. The contents of nitrogen and phosphorus in each group were determined as per the methods described by Ruttenberg (1992) and Ruban et al (2001). The different occurrence forms of nitrogen include an ion-exchanged form (IEF-N), a weak acid extractable form (WAEF-N), a strong alkali extractable form (SAEF-N) and a strong oxidant extractable form (SOEF-N). Different forms of phosphorus include non-apatite inorganic phosphorus (NAIP), apatite inorganic phosphorus (AP), inorganic phosphorus (IP), organic phosphorus (OP), and total phosphorus (TP). IEF-N is the most easily released nitrogen form in sediments, which is mainly released due to the adsorption of nitrate in overlying water and the ammonia nitrogen generated by the decomposition of organic nitrogen in sediments. WAEF-N mainly includes nitrogen bound to CO3^2–, which is affected by CO3^2– concentration in sediments. SAEF-N mainly refers to Fe/Mn bound nitrogen in sediments, and its formation is related to the redox potential in sediments. SOEF-N mainly refers to the organic form of nitrogen in sediments, which is generally the main form of nitrogen. NAIP refers to the phosphorus bound to Fe, Mn, and Al oxides and their hydroxides in sediments, and this form of phosphorus is easily released in the sediments. AP is a form of phosphorus bound to calcium in sediments, which is considered to be relatively inert. IP refers to total inorganic phosphorus in sediments, which mainly includes NAIP and AP. OP refers to organic phosphorus in sediments, and TP refers to all forms of phosphorus in sediments. The results showed that P. aibuhitensis significantly increased the quantity of IEF-N, WAEF-N, and SAEF-N in the upper and middle layers of sediments (P<0.05), and the medium and small size groups significantly decreased the content of SOEF-N in sediments (P<0.05). P. aibuhitensis significantly increased the content of NAIP in the upper and middle layers of sediments (P<0.05), and the medium and small size groups significantly decreased the content of OP in sediments (P<0.05). This study showed that P. aibuhitensis could increase the bottom redox potential and promote the formation of Fe/Al bound nitrogen and phosphorus. It could also accelerate the mineralization of organic nitrogen and phosphorus in sediments, promoting the upward migration of other forms of nitrogen and phosphorus in sediments, making nitrogen and phosphorus in sediments easier to decompose and release, which could be used by phytoplankton in water. In the early stages of pond culture, P. aibuhitensis disturbance can make water nutrient rich in the late stage of culture, P. aibuhitensis can reduce the accumulation of nitrogen and phosphorus in sediments, so that the ponds can maintain good breeding conditions. P. aibuhitensis has the potential to repair environmental pollution in conjunction with other aquatic plants and algae.

Abstract:Black rockfish (Sebastes schlegelii) is a member of the family Sebastidae—an ovoviviparous marine fish widely distributed in the coastal waters of China. This species has important commercial value and is also a delicious table fish. It often inhabits areas at the bottom of the sediment in rocky reefs with clear water quality. The species does not exhibit a long-distance migration habit or remarkable clustering. In recent years, sea ranching has developed rapidly in China. Black rockfish is a good candidate to promote sea ranching, because it does not exhibit a long migratory habit and is called a housekeeping fish. In recent decades, owing to the overfishing of wild resources and deterioration of the environment, the natural pools of black rockfish have decreased sharply. Thus, the national black rockfish release and breeding plan was implemented in 2006. Currently, millions of young black rockfish are released into the sea every year in China, which has played a positive role in promoting the conservation and restoration of natural resources. However, the release activity of marine organisms is random and under lax supervision. In recent years, various tagging technologies, including external markers, such as T-bar markers, and internal markers, such as otolith markers, have been developed to support the evaluation of the enhancement effect of released species. At present, otolith fluorescent labeling is the only reported marker technology that can be used for the proliferation and release of black rockfish. However, otolith staining has some problems, such as difficulty in detection and rapid fading, and it is not effective as an internal marker. Currently, there has been no efficient internal marker for black rockfish juveniles; thus, an effective marking technology must be established for evaluating the enhancement effect of black rockfish to support scientific release and natural resource maintenance. The coded wire tag (CWT) is a magnetized stainless-steel wire segment with a diameter of 0.25 mm. Several rows of numbers are engraved on the marking line to represent a specified batch or a single code. CWTs are widely used internal tags with many advantages, including small size, easy management, accuracy, and high retention. CWTs have been effectively used in over 30 fish genera worldwide because of their high retention rates and minimal biological impact on living fish, although serious effects on growth and survival have been reported among different species and tag placements. In the present study, we investigated the marking effects of CWTs on released juveniles of black rockfish. According to different sizes and marking placements, the experimental juveniles of black rockfish were divided into four groups: small fish [average total length = (8.25±0.84) cm, average body weight = (9.99±2.75) g ] with gill levator arcus palatine muscle tagging group (SLM group); small fish with dorsal muscle tagging group (SDM group), large fish [average total length = (11.13±0.67) cm, average body weight = (20.95±2.99) g ] with gill levator arcus palatine muscle tagging group (SDM group); and large fish with dorsal muscle tagging group (LDM group). The survival rate, tag retention, antioxidant enzyme activity, and expression of genes related to growth or stress were measured to evaluate the effects of CWTs on black rockfish juveniles. The survival rate was 100% in all study groups. The tag retention rates in the SLM and LLM groups were 87% and 97%, respectively. The tag retention rate in the SDM and LDM was 100%. The specific growth rate of all tagged fish was lower than that of the control fish. After 30 days of culture post-tagging, the liver catalase enzyme activity in the SLM and LDM groups was significantly higher than that in the control group, and the liver superoxide dismutase enzyme activity in all experimental groups was significantly higher than that in the control group. The liver IGF-I mRNA levels were significantly higher in the SLM, LLM, and LDM groups than in the control group. There were no significant differences in HSP70 mRNA levels between the small fish groups, while in the large fish groups, HSP70 mRNA level in the LDM group was lower than that in the LLM and control groups. Therefore, CWT is suitable for marking black rockfish juveniles based on survival and tag retention data; however, the specific growth rate of tagged fish was lower than that of the control fish, and the antioxidant enzyme activities and expression of genes related to growth and stress showed significantly different expression responses to CWT marking, indicating that tagging caused physiological stress in released black rockfish juveniles. Therefore, indoor temporary breeding or wild domestication time should be relatively long for the seedlings marked with CWTs, which is conducive to improving the survival rate of the marked released seedlings and their growth after release. Overall, CWTs may be useful to evaluate the enhancement effects of released species and support accurate assessments of the proliferation and release effect of black rockfish.

Abstract:Interferon regulatory factor (irf7) is an immune regulatory factor that plays an important role in the antiviral process. To explore the role of irf7 in Oplegnathus punctatus (Temminck & Schlegel, 1844) under viral infection, we cloned the coding DNA sequence (CDS) region of irf7 through PCR and analyzed the expression patterns at both tissue and cell levels. The results showed that the CDS region of Opirf7 was 1 332 bp and encoded a peptide with 443 amino acids. The predicted molecular weight was 50.5 kDa and the theoretical isoelectric point was 5.546. Protein structure analysis showed that Opirf7 has three conserved domains: the DNA binding domain (DBD), IRF-associated domain (IAD), and serine-rich domain (SRD). Amino acid similarity analysis showed that OpIRF7 had the highest similarity to the IRF7 of Lates calcarifer, which was 82.92%. The similarity of Opirf7 with the IRF7 of Larimichthys crocea, Paralichthys olivaceus, and Cynoglossus semilaevis were 81.99%, 79.95%, and 73.74%, respectively. Phylogenetic analysis showed that Opirf7 and other fish irf7 genes were clustered into one branch, and irf7s from Gallus gallus, Mus musculus, Macaca mulatta, and Homo sapiens were clustered into another. Tissues from healthy O. punctatus were collected, including the liver, spleen, kidney, head kidney, intestine, gill, skin, muscle, brain, heart, and blood. After RNA extraction and cDNA synthesis, real-time PCR (qPCR) was performed to detect the expression level of Opirf7 using the comparative CT method (2−ΔΔCT method). The results of qPCR showed that Opirf7 was expressed in different tissues of healthy individuals and its expression was highest in the liver, followed by the skin and intestines. The lowest expression was observed in the head kidney. In this study, the expression profiles of Opirf7 before and after viral infection were determined at the tissue and cell levels. For the in vivo challenge study, fish were intraperitoneally injected with spotted knifejaw iridovirus, and the expression level of Opirf7 was tested in the spleen, kidney, and liver. Compared with the control group at 0 h, the expression level of Opirf7 was 15-fold in the spleen and 3-fold in the kidney 4 days after infection, and the expression peak was at 7 days after infection. However, the expression of Opirf7 was not significantly altered in the liver. A poly I:C-infected O. punctatus brain cell model was established, and the expression profiles of Opirf7 mRNA were detected before and after infection. The expression of Opirf7 mRNA in the low and medium concentration groups (50 μg/mL and 100 μg/mL, respectively) increased by 13 to 17 times, and the expression level of Opirf7 mRNA in the high concentration group (200 μg/mL) increased by approximately 8 times. It was speculated that the high concentration of 200 μg/mL caused some damage to the cells and that the expression level in the high concentration group was lower than that in the low and medium groups. In this study, the full-length open reading frame sequence of Opirf7 was cloned and characterized for the first time. The deduced amino acid sequence displayed a structure similar to those of other vertebrates. Further functional analysis showed that Opirf7 has a significant response to viral infection at both tissue and cell levels. This study demonstrated that the Opirf7 gene might play an important role in the antiviral response of O. punctatus and provide a potential molecular marker for antivirus breeding of O. punctatus.

Abstract:Environmental conditions regulate the growth and reproduction of fish. The increase in sea temperature during winter may have adverse effects on Takifugu rubripes. To study the mechanism of low-temperature tolerance of T. rubripes, the expression of antifreeze protein (AFP) gene, cold-induced RNA binding protein (CIRP) gene, high mobility group protein box-1 (HMGB1) gene, and Y-box binding protein (YB-1) gene in the liver, spleen, kidney, brain, heart, intestine, muscle, gonad, and skin tissues of T. rubripes obtained from different temperatures (18℃, 13℃, 8℃, and 5℃) was analyzed by quantitative real-time PCR. The results showed that the AFP gene was widely expressed in tissues, with the highest expression in the muscle (P<0.05). With the decrease in temperature, the expression of the AFP gene in each tissue showed a significant increasing trend, reaching the highest value in the 5℃ group. The expression of the CIRP gene was the highest in the muscle (P<0.05). With a decrease in temperature, the trend of CIRP gene expression in various tissues was different. The CIRP gene expression levels of liver, kidney, brain, heart, intestine, and skin showed a trend of initial increase, followed by a decrease, and then an increase. The expression levels in the spleen, muscle, and gonads showed an upward trend, reaching the highest value in the 5℃ group. The expression of the HMGB1 gene was the highest in muscle (P<0.05), followed by that in the brain, liver, heart and skin. As the temperature decreased, the expression of the HMGB1 gene in all tissues except the liver increased first and then decreased, and reached the maximum value in the 8℃ group, which was significantly higher than that of the other groups (P<0.05). The expression of the YB-1 gene was the highest in the muscle (P<0.05), with the lowest expression level in other tissues. As the temperature decreased, the expression level of most tissues (brain, heart, intestine, kidney, liver, muscle, and spleen) increased first, then decreased, and then increased, reaching the minimum value in the 8℃ group (P<0.05). These results show that the expression levels of the four genes are different at different temperature, reflecting the functional specificity of these four genes. Under low-temperature stress, these genes responded positively. Their expression changed to varying degrees, suggesting that the four genes may have potentially important roles in the adaptation of T. rubripes to low temperatures. In addition, by analyzing the law of gene expression, 8℃ may be the key regulatory point for T. rubripes to deal with low-temperature stress. Too low temperature may cause its regulation disorder. The results of this study can provide a relevant basis for studying the regulation mechanism of the low-temperature response of T. rubripes.

Abstract:Fish blood tends to coagulate due to its specialized characteristics. In general, the detection of blood physiological indices using anticoagulants reflects normal physiological status. However, the selection and application of anticoagulants have no uniform standards because of the specificity and diversity of fish species. In the present study, the effects of three different anticoagulants (sodium heparin, K2EDTA, and sodium citrate) were investigated on coagulation, blood cell type, hematological parameters (white blood cells, red blood cells, hematocrit, and hemoglobin), and plasma cortisol and glucose contents in turbot during acute hypoxic stress. The choice of anticoagulants may result in significant differences in turbot blood physiology and chemistry. To supply technological support in turbot hematological research and best-practice aquaculture, this study compared the effects of three common anticoagulants between normal dissolved oxygen and acute hypoxic states. The recirculating aquaculture system is the main culture model for turbot; water temperature and dissolved oxygen levels are important environmental factors, especially in high-density industrial systems. Sufficient oxygen is key to maintaining normal metabolism in turbot. Experimental procedures were designed for control and treatment groups, each group had three parallels, and 54 turbots were studied in all. In the control group, 200 mg/L MS-222 was used to anesthetize a specimen before collecting 5 mL of blood from the caudal vein; the blood was transferred through a needle and vacuum tubes containing three different anticoagulants, then the vacuum tubes were stored for evaluation after 6 h and 12 h. In the treatment group, nitrogen gas was used to rapidly reduce the concentration of dissolved oxygen in the container to (1.2±0.3) mg/L (measured with a dissolved oxygen meter), after which the same procedure of anesthesia and blood collection was conducted. For blood cell type and morphology studies, the Giemsa staining method was used to make blood smears. The original solution of Giemsa was diluted before use. The slide was sterilized before a drop of blood was placed on one side of the slide; one edge of a cover glass was placed in contact with the drop of blood and pushed to the opposite side of the slide at a constant velocity; the slide was then fixed with methanol for 10~15 min and stained for 15~30 min. The smears were washed and dried after staining. Finally, the stained smears were observed under a Leica microscope to determine blood cell morphology, and images were taken. Blood samples were divided into two parts for detecting physiological and biochemical indices: one was centrifuged at 3500 r/min for 10 min, and the supernatant was stored at –80℃ for the detection of plasma glucose and cortisol content using commercial kits. The residual blood samples were stored in a refrigerator at 4℃ for the detection of physiological indices using an automatic blood cell analyzer. The anticoagulant effect of K2EDTA was most effective under a normal dissolved oxygen state, and the anticoagulant effect of K2EDTA and heparin sodium were most effective after 6 h and 12 h of exposure to acute hypoxia stress, respectively. In addition to coagulation, blood cell morphology showed that binucleate cells occurred through sodium heparin, eosinophils, and basophils through sodium citrate and basophils through K2EDTA in the control. Three nucleus abnormality types were identified in the smears: micronucleus cells, binucleate cells, and erythroplastids. Binucleate cells were found using sodium heparin in the control group. In addition, binucleate cells were observed under three different anticoagulants in the treatment group, and micronucleus cells and erythroplastids were observed in the treatment group. The hematological index showed that the number of white blood cells was significantly increased by treatment with three anticoagulants during acute hypoxic stress (P<0.05), and the number of red blood cells and hemoglobin content was significantly increased by treatment with K2EDTA and sodium heparin (P<0.05). However, they were significantly reduced after treatment with sodium citrate. In addition, the plasma glucose and cortisol content were significantly increased when subjected to acute hypoxic stress, but the content of sodium heparin was significantly lower than that of both sodium citrate and K2EDTA. In summary, K2EDTA showed less blood coagulation than other anticoagulants, sodium heparin caused binucleate cells and decreased plasma glucose and cortisol, and sodium citrate affected the number of red blood cells and hemoglobin content. K2EDTA is the more promising anticoagulant compared to sodium heparin and sodium citrate for blood analysis of turbot and promotes the precision of turbot hematological studies under acute hypoxic conditions.

Abstract:Yadong trout (Salmo trutta fario), the only salmonid fish in the Tibetan Plateau, is a national agricultural product with geographical indications in Yadong. The development of Yadong trout aquaculture has significant implications for local farmers. However, no study has yet investigated the nutrient requirements of this fish. This study aimed to analyze the proximate amino acid, and fatty acid composition of various tissues of wild and farmed Yadong trout, in order to provide a reference for the evaluation of fish fillets and the formulation of optimal feed. Ten wild fish captured in the Yadong River, and 10 fish farmed with formulated feed in Yadong Industry Park were used in this study. The farmed fish were F1 generation wild broodstock captured in the Yadong River and fed formulated feeds throughout their lifetime. The body length of wild Yadong trout was in the range of 13.4~28.7 cm, and the body weight was in the range of 38.48~346.85 g. The body length and body weight of farmed Yadong trout were in the range of 16.4~24.5 cm and 78.39~275.29 g, respectively. Frozen samples were transported to the laboratory by flight. The proximate composition of whole fish, muscle, and liver; fatty acid composition of whole fish and various tissues including muscle, liver, intestine, brain, and eye; as well as amino acid composition of whole fish, muscle, and liver were analyzed. The fatty acid composition (expressed as % total fatty acids) and amino acid composition (expressed as % dry matter basis) were assayed using gas chromatography and an automatic amino acid analyzer, respectively. In addition, t-tests were performed for independent samples; results are expressed as the mean ± standard deviation. The results showed that wild fish had lower condition factors but higher moisture and ash content than farmed fish. Wild fish muscle had lower crude lipid content but higher moisture content than farmed fish. The whole-body protein and lipid contents, as well as the proximate composition of the liver, were not significantly different between wild and farmed fish (P>0.05). The contents of saturated fatty acids (mainly C16:0 and C18:0) and n-3 polyunsaturated fatty acid (PUFA) in whole fish, muscle, and liver were significantly higher (P<0.05), whereas contents of monounsaturated fatty acids (MUFA) (mainly C16:1n-7 and C18:1n-9) and n-6 PUFA (mainly C18:2n-6) were significantly lower in wild Yadong trout than in farmed Yadong trout (P<0.05). EPA and C20:4n-6 in whole fish, muscle, and liver, as well as DHA in the muscle of wild Yadong trout were significantly higher than those in farmed Yadong trout (P<0.05). In the intestine of wild Yadong trout, the contents of C14:0, C16:0, C18:0, C16:1n-7, C20:5n-3, and C22:5n-3 were significantly higher (P<0.05), while those of C18:1n-9 and C18:2n-6 were significantly lower (P<0.05) than in the farmed fish. In the brain of wild Yadong trout, the contents of C16:1n-7, C20:5n-3, and C22:5n-3 were significantly higher (P<0.05), while the contents of C18:1n-9 and C20:2n-6 were significantly lower (P<0.05) than those of farmed fish. In the eyes of wild Yadong trout, the contents of C14:0, C16:0, and C18:0, C16:1n-7, C20:1n-9, C20:2n-6, C20:4n-6, C20:5n-3, and C22:5n-3 were significantly higher (P<0.05), while the contents of C18:1n-9, C22:1n-9, C24:1n-9, C18:2n-6, and C18:3n-3 were significantly lower (P<0.05) than those of farmed Yadong trout. The total essential amino acid content in wild fish was significantly higher than that in farmed fish. The contents of threonine, valine, phenylalanine, lysine, and glycine in the muscle of wild fish were significantly higher (P<0.05) than those in farmed fish, while no significant difference (P>0.05) was observed in other amino acids. These results indicate that wild Yadong trout were leaner than farmed trout in terms of muscle lipid content. For trout, which are mainly consumed fresh, this trait may make farmed Yadong trout more acceptable than wild trout. However, in terms of fatty acid composition and amino acids, the wild Yadong trout seemed to be more acceptable than farmed trout. In particular, n-3 PUFA content is an important nutritional trait in fish fillets. The wild Yadong trout had higher n-3 PUFA contents than the farmed trout. Thus, the fish oil in the feeds of Yadong trout should not be omitted, considering that fish chemical composition generally reflects that of their diets. In addition, the price of alternative oils such as soybean oil is no longer low; the use of lipid sources in the diets of Yadong trout should be reviewed. Similarly, the essential amino acid content in wild Yadong trout was higher than that in the farmed trout, suggesting that the protein sources should be reviewed, and a certain level of fish meal should be guaranteed. In conclusion, the results of this study indicate that the formulated feeds of Yadong trout need to be further optimized.

Abstract:Temperature is an important environmental factor that affects the growth, development, and metabolism of shrimp. High temperatures tend to cause a decrease in the immunity and production performance of shrimp, resulting in a significant increase in shrimp disease and mortality. Based on transcriptome sequencing data, we screened for the involvement of DNA damage-inducible transcript 4 like (ddit4l) in the temperature stress process of Litopenaeus vannamei. Ddit4l (also known as Redd2 and Rtp801L) is involved in regulating cell survival, proliferation, and apoptosis by affecting DNA damage repair factors, hypoxia-inducible factors, and other signaling pathways when stimulated by DNA damage, hypoxia, ischemia, and oxidative stress, among others. Ddit4l may be an important transducer of pathological stress in autophagy through mammalian target of rapamycin (mTOR) signaling. Autophagy is a process in which cells use lysosomes to degrade their own damaged organelles and macromolecules, and the process plays an important role in maintaining the balance of protein metabolism and the stability of the intracellular environment, promoting cell growth and development. An important regulator of autophagy is the mTOR, which responds to various stimuli, such as growth factors, cellular energy status, oxygen concentrations, and stress, to regulate cell metabolism and growth. Ddit4l and its homolog ddit4 are upstream mTOR inhibitors in several tissues and cell models. We set up sudden temperature change and gradual temperature change experiments to induce a stress response in L. vannamei. In the gradual change experiment, the water temperature was increased from 26℃ to 38℃ at a rate of 4℃ per day, and cooled back to 26℃ at the same rate. Two individuals were randomly selected from each of the three parallel experiments in the experimental group at each time point. The eyestalk, gill, hepatopancreas, nerve, and muscle were used for subsequent RNA extraction and gene expression quantification. RNA quality and concentration were also examined. The partial ddit4l cDNA (complete ORF and partial UTR) of L. vannamei was obtained directly by PCR amplification to evaluate the structure and function of the Lv-ddit4l gene. The Lv-ddit4l sequence contained a 495 bp open reading frame, encoding 164 amino acids, with a molecular weight of 18.51 kDa, and an isoelectric point of 5.35. The instability index was 65.83, which classifies the protein as unstable. Lv-ddit4l contains a highly conserved RTP801-C domain. BLAST alignment and phylogenetic tree analysis showed that the Lv-ddit4l gene had 59.76% similarity with the Chionoecetes opilio ddit4l gene, and first clustered with C. opilio. A real-time RT-PCR confirmatory experiment considered three parallel groups for each sample with 18S rRNA as the reference gene. Relative gene expression was analyzed using the 2–ΔΔCt method. Quantitative analysis of gene expression levels showed that Lv-ddit4l was widely expressed in all of the examined tissues, with similar expression levels at 26℃. When the temperature was increased gradually from 26℃ to 32℃, the Lv-ddit4l gene in the eyestalk was significantly increased (P<0.05), and was 10.37 times higher than that in the control (A-26℃). Therefore, this gene is hypothesized to respond to temperature changes in the eyestalk of L. vannamei. The expression of Lv-ddit4l in the eyestalk, gills, hepatopancreas, nerves, and muscles did not change significantly when the water temperature was gradually increased to the highest temperature of 38℃; it is speculated that this gene may play a role in pre-heat stress. When the temperature was gradually cooled from 38℃ to 32℃, the expression of Lv-ddit4l increased significantly in the gill and hepatopancreas (P<0.05), which were respectively 3.37 and 2.03 times higher than that of the control group (A-26℃). There was no significant difference in gene expression under different levels of gradual changes in temperature stress in the nerve and muscle. The expression in the gill, hepatopancreas, and nerve was significantly up-regulated (P<0.05) at a sudden change in water temperature to 36℃ for 10 min, and the change in expression in the hepatopancreas was 24.06 times higher than that in the control group (A-26℃). Gene expression in the gills and nerves was 1.22-fold and 3.67-fold higher than that in the control (A-26℃), respectively. When the water temperature was increased to 36℃ suddenly for 24 h, Lv-ddit4l gene expression increased significantly in the muscle (P<0.05). Lv-ddit4l showed significant up-regulation of expression in the gill, hepatopancreas, nerve, and muscle with sudden changes in temperature, suggesting that this gene may play a role in the stress induced by sudden increases in temperature. When the temperature was restored from 38℃ and 36℃ to 26℃, the expression of the Lv-ddit4l gene was downregulated in L. vannamei. The above results indicate that Lv-ddit4l is associated with the response to high-temperature stress.

Abstract:Dissolved oxygen (DO) is one of essential factor for the survival of aquatic organisms, affecting many physiological processes including growth, survival, reproduction and development. Due to the change of global environment and the influence of human activities, hypoxia often occurs in both natural and cultured water bodies. Studies showed that water is in a hypoxic state when DO is lower than 2 mg/L, and aquatic animals in this condition may often suffer from hypoxic stress. The blood clam Scapharca broughtonii is an important economic bivalve because of its delicious taste and rich protein, vitamins and hemoglobin. At present, the S. broughtonii has become an important species in Northern China, and the better economic benefit of breeding drives the rapid development of seedling production and culture. However, hypoxia often threatens the cultivation of S. broughtonii. Hypoxic preconditioning refers to the endogenous protective mechanism generated after the body is stimulated by multiple transient and non-fatal hypoxic stimuli, which can protect the body tissues and improve the tolerance of the body. To explore the effects of hypoxic preconditioning on the physiological and biochemical characteristics of S. broughtonii under hypoxia stress, the clams were divided into three groups containing H2 (hypoxic preconditioning for two times), H4 (hypoxic preconditioning for four times) and C (no hypoxic preconditioning, as control group). And then, we analyzed the feeding rate (IR), energy metabolism and three enzymes activity of three groups during the process of hypoxia stress for 48 hours with DO concentration of ~2.0 mg/L. The first item we explored was changes in feeding rates. During hypoxia stress, the IR of the three groups showed a tendency of decreasing first and then increasing. At the late stage of stress, the feeding rate of the three groups recovered to some extent, and the recovery degree of groups H4 and H2 were better than that of group C. The energy metabolism we studied contains the oxygen consumption rate (OR) and the ammonia excretion rate (NR). The results showed that the OR of the three groups increased gradually under hypoxia stress and reached the maximum value at 48 h after treatment. The OR of group C, H2 and H4 increased by 1.15, 1.08 and 0.73 times, respectively, after 48 hours of treatment. The trends in NR were that the NR in group C gradually increased during treatment, while that in group H reduced first followed by increasing and then decreased. The NR of group C, H2 and H4 showed different trends during the stress period which were 1.67, 1.30 and 0.97 times of 0 h at 48 h, respectively. And the ratio of oxygen to nitrogen in group C was relatively stable, while that in group H was relatively wide. The following are the experimental results of enzyme activity changes, which include cytochrome c oxidase (COX), lactate dehydrogenase (LDH) and glutathione (GSH). The COX of the three groups decreased gradually with the extension of hypoxia stress time. LDH activity and GSH enzyme content all showed an upward tendency. Compared with the control group C, the enzyme activity of the pre-hypoxic group H2 and H4 were relatively stable during the hypoxic stress. In total, the results showed that S. broughtonii pretreated with hypoxia had higher feeding rate, lower oxygen consumption rate and relatively stable enzyme activity compared with the control group, which revealed hypoxia pretreatment could improve its ability of hypoxia tolerance. It showed that hypoxia preconditioning had a more positive effect on the hypoxia tolerance of S. broughtonii. In this study, the effects of hypoxic preconditioning on physiological and biochemical of S. broughtonii under hypoxia stress including feeding rate, respiratory metabolism and enzyme activity were studied for the first time. It was found that hypoxic training could effectively improve the body's response to hypoxic stress, enriching the research data related to hypoxic adaptation, and providing data for exploring the stress adaptation mechanism of S. broughtonii.

Abstract:microRNAs (miRNAs), involved in post-transcriptional gene regulation, play important roles in the growth, development, cell differentiation, and immune defense of eukaryotes. Apostichopus japonicus has become an economically important species for marine aquaculture in China; however, the outbreak of diseases, such as skin ulcer syndrome (SUS), has led to great losses in aquaculture development. Therefore, molecular mechanisms underlying disease occurrence must be further elucidated. In the present study, Vibrio splendidus, an important causative pathogen of SUS, was used as the stress strain (1×106 CFU/mL) in an artificial infection experiment. Body wall of the diseased (PT16S) and healthy (PT10H) samples was subjected to miRNA-Seq. Differentially expressed miRNAs (DEmiRNAs) were screened using bioinformatics. The target genes of DEmiRNAs were predicted and used for constructing miRNA-mRNA regulatory networks. Through miRNA-Seq, respectively 5 902 588 and 5 053 529 valid reads were generated for the PT10H and PT16S samples. A total of 13 DEmiRNAs (P≤0.05) were screened between PT10H and PT16S, of which two were upregulated and 11 downregulated. Target gene prediction revealed that the two upregulated DEmiRNAs bound to 3010 target genes, which were annotated to 585 GO terms and 24 signaling pathways (P≤0.05), while the 11 downregulated DEmiRNAs bound to 19 072 target genes, which were annotated to 514 GO terms and 22 signaling pathways (P≤0.05). In the validation test, the consistency rate of the sequencing and qRT-PCR data reached 70%. Two immune-related pathways (ubiquitin-mediated proteolysis and Notch signaling) were selected and used to construct the miRNA-mRNA regulatory networks. The 13 DEmiRNAs could bind 134 ubiquitin-mediated proteolytic mRNAs and 109 Notch signaling mRNAs. Specifically, Aja-miR-1559-3p, Aja-miR-7550-5p, Aja-miR-2478, and Aja-miR-9277-3p may be involved in the regulation of ubiquitin-mediated proteolysis and Notch signaling. Our results provide primary data for understanding the post-transcriptional regulatory mechanisms of diseases in sea cucumber.

Abstract:Macrocystis pyrifera is a large perennial brown alga used as a raw material in the chemical, energy, and medicine industries. It is also a high-quality material for the construction of seaweed beds with extremely high economic and ecological value. In the 1980s, M. pyrifera was introduced to China, and many experiments on its seedling and cultivation technologies were undertaken. However, research on its ecological adaptability is relatively challenging, and the aquaculture industry has not yet developed due to bottleneck problems. In the present study, the MaxEnt model was used to predict the suitability and potential invasion risk of M. pyrifera in China to lay a foundation for M. pyrifera culture industry development and marine ecological construction. Parameter optimization showed that the predictive performance of the model was the best when the feature combination was product, quadratic, and hinge features and the regularization multiplier was 0.8. Considering the correlation between environmental attributes and their contribution to the model, six environmental factors were selected to construct a prediction model for the suitability of M. pyrifera. Among these, light intensity and temperature produced the greatest impact on the natural distribution of M. pyrifera. For high suitable growth probability, the optimal light intensity was > 2 μmol/(m2·s) and the optimum temperature range was 10.5~17℃. Combined with ArcGIS, the modeling results showed that the suitable habitats for M. pyrifera in China are mainly distributed in the Yellow Sea and Bohai Sea, accounting for approximately 13.17% of the sea area, with a marginal suitability of 5.46%, low suitability of 2.85%, moderate suitability of 1.20%, and high suitability of 3.66%. Furthermore, Liaodong Bay and Bohai Bay are suitable sea areas for the introduction and cultivation of M. pyrifera as well as the construction of M. pyrifera farms. Some areas in Liaodong Bay are highly suitable, indicating a certain risk of invasion. Therefore, ecological safety evaluations should be strengthened if M. pyrifera cultivation is promoted near this area.

Abstract:Giant kelp (Macrocystis pyrifera, Laminariales), has long been considered one of the most promising macroalgal species for biomass production because of its large size, rapid growth rate, and dynamic life history strategies. Brown seaweeds are economically important and commonly used for agricultural and industrial purposes. Intertidal and subtidal habitats, which most brown algae inhabit, are shaped by fluctuating levels of salinity-, temperature-, and light-related stresses. The responses of brown algae to abiotic stress have been comparatively well studied. With the rapid development of modern industry and agriculture as well as the exploitation of mineral resources, the pollution of ecological environments, particularly heavy-metal contamination of water, is becoming increasingly serious. Natural quantities of heavy metals in seawater do not adversely affect marine life, and some heavy metals even serve as trace nutrients essential for the normal growth and metabolism of algae. However, at excess concentrations, heavy metals act as pollutants and harm algae, and the magnitude of their impact varies depending on the degree of pollution. High metal concentrations negatively affect diatoms by inhibiting growth, triggering oxidative damage, modifying gene expression, damaging photosynthetic cells and mitochondria, and disrupting various cellular processes. Among the various metals, cadmium is particularly toxic and can easily accumulate in many marine organisms. Usually, cadmium concentrations in the sediment and open seawater are low, although these values may increase in some offshore and estuarine areas due to leakage or anthropogenic emissions. Glutathione S-transferase (GST) is a phase Ⅱ enzyme in cells that catalyzes the formation of chelates between reduced glutathione and metal ions as well as the binding of sulfur atoms of reduced glutathione to phase Ⅰ electrophilic groups, thereby reducing the levels of intracellular toxic substances, such as reactive oxygen species, and accelerating their exogenous release. GST belongs to a large gene family that plays important regulatory roles in growth, development, and responses to environmental fluctuations. Owing to the lack of a stable genetic operating system in M. pyrifera, the functions of some genes and proteins remain unclear. To date, there has been no successful genetic transformation of M. pyrifera. Synechococcus elongatus PCC7942 is easy to culture, has a small genome size, and can easily be genetically manipulated through natural transformation or conjugation with Escherichia coli, making it a good protein expression system for studying prokaryotic circadian rhythms, nutrient regulation, environmental responses, and lipid metabolism. In the present study, S. elongatus PCC7942 was selected to verify the functions of the GST gene in M. pyrifera under Cd stress. Total RNA was extracted from M. pyrifera gametophytes frozen in liquid nitrogen and reverse-transcribed to cDNA. Gene-specific primers containing enzyme restriction sites at both ends were designed to construct an expression vector based on the transcriptome sequence of M. pyrifera (accession number CNP0001061 in China National GenBank). Six complete GST genes (mpgst1, mpgst2, mpgst3, mpgst4, mpgst5, and mpgst6) were cloned using RT-PCR. Subsequently, the six MPGST genes were transformed into S. elongatus PCC7942, and the transformed strains containing mpgst1–mpgst6 were labelled MG1–MG6, respectively. Transformation was verified by genomic DNA extraction and GST enzyme activity assays. In this study, 0.2 mg/L was determined as the Cd2+ concentration that was lethal to the wild strain but enabled normal growth of some transformed strains. Some of the transformed strains did not exhibit resistance, which may be due to differences in the GST gene sequences of M. pyrifera or because they may belong to different GST gene families, serving different functions. The selected resistant transformed strains MG1, MG4, and MG6 were tested for growth, photosynthetic pigment content, and photosynthetic parameters at 0.2 mg/L cadmium ion stress to verify their functions. The transformed strains showed an upward trend of light absorbance, but most of the wild type strains died. Furthermore, the transformed strains presented values for photosynthetic pigment content and photosynthetic parameters even under stress, but the wild strain died, which was consistent with the growth curve. In particular, the carotenoid content of MG6 slightly increased following Cd2+ stress, indicating elevated antioxidant activity. However, differences in the physiological indices of different genes before and after stress may be related to their specific mechanisms of action, which warrants further study. Our findings laid a foundation for further research into the stress resistance function of GST genes in M. pyrifera and for the future breeding of pollution-tolerant algal strains.

Abstract:Neoporphyra haitanensis is a traditional coastal aquaculture species in China; it is rich in essential amino acids, minerals, and vitamins. It is not only beneficial economically, but also adjusts the ecosystem. However, fine varieties of N. haitanensis are lacking even with the enlargement and extension of the N. haitanensis growing area. Since humans began breeding N. haitanensis, most produced strains have been domesticated from wild N. haitanensis collected from rocks. The germplasm collection and usage is considered the bottleneck issue that often causes N. haitanensis quality degradation. Therefore, it is important to breed new varieties. Selective breeding, cross-breeding, and mutation breeding are among the most common methods used to breed new N. haitanensis varieties. Mutation breeding is a quick, simple, and convenient genetic tools. For several decades, natural means to induce genetic diversity have been exploited to breed new varieties, as the naturally occurring mutations are insufficient. One of the most important genetic breakthroughs was the invention of artificial methods to induce mutations. Physical mutagenesis is a safer and more efficient procedure than chemical mutagenesis. To obtain artificial mutants, N. haitanensis (NSD35) gametophytes were treated with different γ-ray irradiation intensities (700 Gy, 900 Gy, 1100 Gy, 1300 Gy, and 1500 Gy). After culture recovery, the results showed that gametophytic cell death increased with the irradiation escalation, with more than 90% cell viability after low radiation dose treatment (700 Gy and 900 Gy). With doses more than 1100 Gy, the cell viability decreased, in which the NSD35 cellular space became larger and a few middle cells died, although no obvious change was observed in tip cells. After induction, the cell morphology changed with higher radiation doses (1300 Gy and 1500 Gy). Some cells exhibited microscopic features, such as enlarged size and alternation from irregular cell shape to circular for some cells. Notably, the cell death rate decreased systematically from the blade base to its tip and from that to its center. Microscopic examination revealed that the number of mutant cells increased and then decreased with the radiation dose rise. The pigmented mutant cells frequency was higher in low radiation doses, further increasing with the rise in irradiation from 700 Gy to 1300 Gy. Nonetheless, the pigmented mutant cells frequency decreased with the highest dose of 1500 Gy. The best mutagenic effect was observed in gametophytes treated with 1300 Gy as they had the largest number of pigmented mutant cells. Cells cultured in vitro were obtained by enzymatic hydrolysis and individual regeneration. Preliminary screening of 67 mutants was performed to construct the mutant library, and 45 pigmented mutant strains, 51 morphological mutants, and 11 other mutants were obtained. Most of the mutants showed two or more mutations of phenotypic character. Twenty-one F1 generation cell lines were screened, and 14 traits (length, width, fresh weight, daily length growth rate, daily width growth rate, daily weight growth rate, length-breadth ratio, base thickness, center thickness, tip thickness, color, base section form, leaf form, saw tooth size, twist, and maturity) were analyzed by correlation analysis and system analysis. The coefficients of variation results showed significant phenotypic differences between F1 and control cells. The F1 coefficients of variation ranged from 8.74% to 59.49%, revealing a moderate variability. Correlation analysis also showed that most of the traits had significant correlations. There was no significant correlation between the length and width or fresh weight, while a significant positive correlation was observed between the width and leaf fresh weight (P<0.01). Significant positive correlations were also found between quantitative traits, while there was no correlation between qualitative and quantitative traits. These allow indirect selection to improve breeding efficiency. Cluster analysis showed that at the Euclidean distance of 20, all 21 individuals analyzed were clustered into four groups. Overall, the phenotypic traits of different groups were significantly different. Mutants are critical materials for studying the genetic regulation of mechanisms involved in the control of economically important N. haitanensis traits. Pigmented mutants are a dominant-marker trait used for breeding that have high value for theoretical research and practical applications. Our study identified many pigmented mutants that can be used to study relevant biological mechanisms. Nevertheless, the variation coefficients of four quantitative characters (length, width, fresh weight, and thickness) of the F1 N. haitanensis generation were less than those of the control group and were biased towards negative variation. For example, more thin leaf mutants were obtained after the gamma rays´ treatment. It is interesting as a thin N. haitanensis is more suitable for automatic processing, and its primary products can be reprocessed with higher added value. In conclusion, after being treated with γ-ray, the N. haitanensis blades were guided to variation enabling the selection and cultivation of new strains, from which strains with improved traits strains were selected. This study provides interesting materials for accelerating the N. haitanensis breeding research and the selection of excellent varieties.

Abstract:Sargassum thunbergii is distributed on reefs and rock marshes in mid- and low-tide zones, and some are periodically exposed to lengthy low tides. Dehydration is a key factor affecting the survival of S. thunbergii at low tides. In this study, using wild S. thunbergii as experimental material, the water loss rate, chlorophyll fluorescence parameters, and biochemical parameters under different stresses were determined by dehydrating the thalli in an incubator for 0, 1, 3, and 6 h. The results showed that: (1) algae of different sizes have significantly different water loss rates under different stresses. The shorter the stress time and the larger the algae, the lower the water loss rate, indicating that the water retention capacity of S. thunbergii with larger thalli is higher. Wild S. thunbergii grow in clusters on reefs. The leaves in the lower part of the branches and near the holdfast are wide. The middle and upper leaves are narrow and long, respectively. The lower broad leaves are easily blocked by the upper branches. Therefore, differences in the growth environment cause differences in the ecological structure and biochemical components of S. thunbergii. High temperature, strong light, and water loss at low tide are the main factors that cause severe environmental stress to sessile S. thunbergii in the intertidal zone. (2) Dehydration significantly reduced the chlorophyll fluorescence value of S. thunbergii, and different parts of the same individual of S. thunbergii had significantly different tolerance to dehydration, with the lowest tolerance at branch tips and the strongest tolerance at the base. The non-regulatory energy-dissipation mechanism plays a major role in the dehydration response of S. thunbergii. Under dry exposure, the light energy utilization efficiency of S. thunbergii was significantly reduced. This reduction in active light protection capacity indicates that dehydration reduces the adaptability of S. thunbergii to excessive light intensity. Dehydration can damage the tips of small individuals that cannot recover, while the base part of large individuals could return to a normal physiological state. (3) Antioxidant enzymes (ASAFR, SOD) and non-antioxidant substances (soluble sugar and proline) in the tip part responded to dehydration, and the base part mainly responded by upregulation of protein, soluble sugar, and proline content to resist stress. S. thunbergii, located in the high and middle tide zones, is more likely to be stressed by high temperatures, strong light, and dehydration, and the physiological and biochemical characteristics of different parts of the thallus are also variable due to differences in external morphology. Algae mainly reduce damage to the photosynthetic system caused by a lack of water through a non-regulatory energy dissipation mechanism. The water retention capacity of the base was better than branch tips during dry exposure, and the damage to algal cells was low. The main roles are as heat shock proteins, soluble sugars, proline, and other small molecules, which can pass stress response, osmotic regulation, and anti-oxidation resists damage to cells caused by stress. The water retention capacity of the top cells was weak, and the stress was relatively strong. Antioxidant enzymes such as ASAFR and SOD in algae and non-antioxidant enzymes such as soluble sugars and proline, work together to resist dry exposure stress, reduce cell damage and maintain cell viability. In summary, under the stress of dry exposure, the antioxidant enzymes, antioxidant substances, and non-regulatory energy dissipation mechanisms of S. thunbergii play a role in maintaining cell activity. This study provides important guidance for exploring the ecological adaptability of S. thunbergii in resisting environmental stress.

Abstract:Ningxia Hui Autonomous Region is located in Northwest China. Most of the culture ponds in Shizuishan City and Yinchuan City, in the northern part of Ningxia, are sulfate-type saline-alkali ponds. The central area of Ningxia is located on the Hetao Plain, where the salinity of water bodies is relatively low. Owing to the high pH value of saline-alkali water bodies, which indicates high alkalinity, water quality control has become the main challenge of saline-alkali aquaculture. The community structure of planktonic microalgae is closely related to the water quality of aquaculture ponds and the healthy growth of aquaculture organisms. The planktonic microalgae community structure of culture ponds in summer in Ningxia is rarely reported. To study the community structure characteristics of planktonic microalgae in different culture pond modes in Ningxia in summer, we collected planktonic microalgae samples in July 2021 from Pingluo County in Shizuishan City, Helan County in Yinchuan City, and Shapotou District in Zhongwei City in the Ningxia Hui Autonomous Region. We then analyzed the planktonic microalgae community composition and its correlation with water quality factors. The samples were collected from three typical aquaculture pond modes: the shed-pond relay mode (PT), soil pond mode (TC), and rice-fishing breeding mode (DY). We qualitatively identified and quantitatively counted planktonic microalgae under a light microscope and performed diversity analysis using the Shannon diversity index. SPSS software was used to carry out bivariate correlation analysis between the total number of algae, total biomass, algal biomass of each phylum, and the physical and chemical factors of water quality in aquaculture ponds. A total of 5 phyla and 27 genera of planktonic microalgae were detected, including 7 genera of Cyanophyta, 13 genera of Chlorophyta, 5 genera of Bacillariophyta, 1 genus of Euglenophyta, and 1 genus of Dinophyta. The total number of planktonic microalgae in the 9 ponds ranged from 1.52×104 to 2.39×108 ind./L, the biomass ranged from 0.16 to 97.78 mg/L, the Shannon-Wiener index as 0.03 to 3.31, and the biomass diversity index was 0.29 to 3.58. There were significant differences in the community structure of planktonic microalgae in different kinds of culture modes. The community structure of planktonic microalgae in PT culture ponds had no obvious common characteristics. Cyanophyta, Chlorophyta, and Bacillariophyta, such as Anabaenopsis sp., Anabaena sp., Oscillatoria sp., Pediastrum sp., Oocystis sp., and Cyclotella sp., were predominant. A single genus of planktonic microalgae dominated in TC. The three ponds were dominated by Pediastrum sp., Chlorella sp., and Microcystis sp. The diversity of microalgae in DY culture ponds was relatively rich. Chlorophyta such as Chlorella sp., Scenedesmus sp., Pediastrum sp, Oocystis sp., and Cladophora sp. and Bacillariophyta such as Cyclotella sp. and Nitzschia sp. were the dominant algae. Although the community structure of planktonic microalgae in most ponds was dominated by Chlorophyta and Bacillariophyta, some ponds in PT and TC still had Cyanophyta overgrowth which became the single dominant algae. Harmful Cyanophyta proliferate in large numbers in aquaculture ponds and easily form algal blooms, which cause significant adverse effects on the water quality and environment and endanger the life and health of cultured organisms. The ponds with Chlorophyta and Bacillariophyta as the dominant species had a relatively stable water quality, were rich in microalgae species, had excellent water color, and experienced less disease and better growth of cultured organisms. The characteristics of high summer temperatures, long sunshine hours, sulfate-type saline-alkali water in Ningxia, and the eutrophication of the culture ponds due to factors such as food and cultured biological excrement affect the structure of planktonic microalgae communities in local culture ponds. The biomass of Cyanophyta had a significant positive correlation with the concentration levels of NO3–-N, NO2–-N, and chemical oxygen demand (COD) (P<0.01). The total nitrogen and COD contents of the ponds in the DY mode were lower than those in the PT and TC modes, meaning that the water environment was not conducive to the proliferation of harmful Cyanophyta and that the planktonic microalgae biodiversity index was relatively high in the DY mode. In addition, a large number of studies have shown that the adaptability of Cyanophyta to strong light and optimal growth temperature is generally higher than that of other algae. The results indicated that in Ningxia, due to the long hours of light in summer, cyanobacteria blooms dominated by Cyanophyta such as Microcystis and Anabaenopsis formed easily in aquaculture ponds with high water carbon and nitrogen nutrients. Therefore, this finding indicates that the hours of light should be used as one of the critical control points when regulating pond water quality to prevent and control the occurrence of adverse conditions such as reduced production and efficiency due to the outbreak of harmful algal blooms.

Abstract:Ostreid herpesvirus 1 (OsHV-1) has caused a serious economic loss to the global bivalve aquaculture industry. Given its wide host range and the frequent emergence of mutated strains, OsHV-1 infection threatens mollusk production. Unlike the common vertebrate herpesviruses that generally exhibit high host specificity, more than ten bivalve species have been identified as potentially susceptible to OsHV-1 infection, including members of Ostreoida, Pterioida, Arcoida, and Veneroida. A variety of OsHV-1 detection methods have been developed, such as PCR, in situ hybridization, in situ PCR, histopathology, transmission electron microscopy, ring-mediated isothermal nucleic acid amplification (LAMP), and recombinase polymerase amplification (RPA). The sensitivity and specificity of transmission electron microscopy detection are low, and specific complementary detection methods such as PCR are needed to confirm the occurrence of OsHV-1. In situ hybridization, although highly specific, has the disadvantages of low sensitivity, complicated procedures, and high investment of effort. PCR methods are still the most widely used for epidemiological investigation of OsHV-1. However, PCR cannot alone confirm infection, which has led to the emergence of unconfirmed cases of OsHV-1 infection in many species and regions. According to the World Organization for Animal Health´s (OIE) Aquatic Animal Manual, positive nucleic acid-specific detection combined with histopathology and transmission electron microscopy is required for the confirmed diagnosis of OsHV-1 infection. Other detection methods, such as LAMP and RPA, which use nucleic acid amplification alone to detect OsHV-1, can not confirm infection as the presence of nucleic acids is not equivalent to viral infection. As a result, the susceptibility of several bivalve species to OsHV-1 infection has not been evaluated. Instead, several obstacles remain with regards to the development of epidemiological surveillance programs and the implementation of quarantine, prevention, and control measures for OsHV-1 infection. Compared with traditional in situ hybridization and in situ PCR assays, in situ LAMP has the advantages of low nucleic acid amplification reaction temperature, less damage to tissue, constant temperature amplification that does not require special experimental equipment, and more convenient and efficient experimental design. In this study, we selected a set of LAMP primers designed for specific detection of OsHV-1, and a pair of loop primers were designed to improve the specificity and stability of the LAMP reaction on slides. An optimized in situ LAMP method for OsHV-1 detection was developed, which provides a rapid diagnostic method with high specificity and sensitivity to identify hosts that are potentially susceptible to OsHV-1 infection, and to characterize the distribution pattern and tissue affinity of the virus in new hosts. The optimized protocol and quantitative PCR (qPCR) were then used to detect OsHV-1 infection in bivalve samples (Crassostrea gigas, C. angulata, Chlamys farreri, Mizuhopecten yessoensis, Ruditapes philippinarum, and Scapharca subcrenata) collected from 2019 to 2021. OsHV-1 hybridization signals were observed in S. subcrenata samples alone, although positive qPCR results were obtained in more species. Further investigation of pathological characteristics and associated viral hybridization signals indicated that OsHV-1 infection always occurred in fibroblasts and hemocytes in the connective tissues of the mantle and hepatopancreas, nucleus of muscle cells in the foot, and adductor muscle. Signals were also occasionally observed in infiltrated hemocytes between and within the gill filaments. OsHV-1 hybridization signals were observed within the hemocytes infiltrating several organs of the furcula visceral cluster. The hemocytes of S. subcrenata seem to be particularly susceptible to OsHV-1 infection. Histopathological lesions and viral hybridization signals in the hepatopancreas organ were consistently observed in individuals with clinical signs, and we recommend the hepatopancreas as the preferred target organ for histopathological and in situ LAMP assays for OsHV-1 infection in S. subcrenata. In situ LAMP-based detection indicated that S. subcrenata was a susceptible host for OsHV-1, and the connective tissue, muscle tissue, and hemolymph cells had a strong affinity for the virus. At present, the susceptibility of many cultured and wild shellfish to OsHV-1 infection is still unknown. Since the emergence of mollusk mortalities associated with OsHV-1 infection occurred in China in the 1990s, its host range has expanded and changed along with the environment, which has complicated its prevention and control. In recent years, the scale of artificial breeding and cultivation of triploid C. gigas has expanded in China, and larval mortalities are frequently associated with OsHV-1 infection. The in situ LAMP detection method for OsHV-1 infection developed in this study proved to be convenient and fast and could be a valuable tool for the rapid detection and confirmation of OsHV-1 infection.

Abstract:China is the largest producer of Neopyropia yezoensis, ranking first in the world for cultivation area and yield production. In N. yezoensis production, diseases occur frequently every year due to increased farming density, environmental deterioration, and germplasm degeneration, resulting in serious economic losses to farmers. Red rot disease is caused by Pythium sp. and is one of the most common diseases during N. yezoensis farming, leading to empty nets and harvest loss. Air-dry, cold storage, and acid wash are common methods to counteract red rot disease in N. yezoensis farming. These physical or chemical disinfection methods, however, are not completely effective, and some have serious consequences. For example, refrigeration equipment and space will greatly increase costs, and acid wash treatments can cause environmental pollution. Although research has attempted to select or cultivate disease-resistant strains of laver, there remains no laver strain completely immune to red rot disease. Biocontrol is an effective method that is widely used in disease control of land crops. Biocontrol is potentially an environment-friendly and effective control method for macroalgal diseases. However, limited information exists on biocontrol in macroalgal diseases. During the growth and development of macroalgae, a variety of metabolites are produced on their surfaces, which provide suitable substrates for microbial colonization. The microbial community attached to the surface of algae is highly diverse and can produce many kinds of biologically active compounds. These compounds not only play a major role in normal morphology, growth, and development of algae, but also have antibacterial, antiviral, antiparasitic, and other activities to protect the host from harmful organisms. Therefore, the epiphytic microorganisms of algae provide good sources of microorganisms for biological screening. This study aimed to screen and identify bacteria with antagonistic ability towards Pythium sp.. A total of 385 bacterial strains, isolated from farming algae and their culturing environments, were screened. In the first round of screening, the plate confrontation method was used and repeated twice and confirmed that nine strains had antagonistic effects on the growth of Pythium sp.. The diameter of the bacteriostatic zone was approximately 1.65–16.54 mm. In the second round of screening, three strains (assigned as P3, P6, and P19) were further investigated using the toxic medium method for inhibitory activities in their extracellular products. Repeated experiments showed that the bacteriostatic rate was approximately 20.04%–30.09%. The antibacterial spectrum was determined by the plate confrontation method. Strains P3, P6, and P19 all had antagonistic effects on the eight tested strains of Pythium preserved in our laboratory. The inhibition rates reached 52.09%–97.95% for P3, 26.81%–78.04% for P6, 10.47%–41.91% for P19, respectively. The Pythium hyphae on the confrontation edge were further investigated by lactic acid phenol cotton blue staining. When compared with Pythium hyphae in a control group, the density and color of Pythium hyphae against strains P3 and P19 became sparse and lighter. There were no significant changes in Pythium hyphae against strain P6. Strains P3 and P6 were identified as Pseudoalteromonas piscicida, and P19 as P. peptidolytica, based on 16S rRNA gene identification and multilocus sequences analysis of 16S rRNA-dnaA-dnaN-recA. The bacterial strains of P3, P6, and P19 had significant antagonistic capabilities against the pathogenic Pythium strains. This indicates they are potential biocontrol probiotics for the control of red rot disease in N. yezoensis. The present study provides the foundation for research on the evaluation and application of antagonistic bacterial strains in the biocontrol of red rot disease of N. yezoensis.

Abstract:Rice flower carp (Cyprinus carpio rubrofuscus) has high economic value because of its tender meat. However, a comprehensive scientific evaluation of the meat quality and nutritional value of rice flower carp is still needed. At the same time, because of its fast growth and strong disease resistance, rice flower carp is widely popularized in rice field culture but without any research comparing its quality with the pond culture method. Although rice farming has high ecological value, the fish yield is low and its specifications are abnormal, which cannot guarantee the stability and safety of the commercial fish supply and limit the potential rice flower carp industrial benefits. In addition, some studies have shown that fish muscle quality can be affected by environmental conditions. This study aimed to comprehensively evaluate the muscle quality and nutritional value of the rice flower carp and investigate the relationships between the nutritional values and its culture conditions, providing data to increase the rice flower carp yield, economic efficiency, and farmed varieties available. Therefore, 6 000 Quanzhou rice flower carp (2.35±0.08 g) were randomly divided into ponds and rice field groups for the experiment (three replicates per group, with a density of 15 000/hm2). The pond culture group was fed with 3% of the commercially established everyday food per fish weight, while the rice field group was not fed. After 12 weeks, the fish were submitted to a 24 h period without food and anesthetized using MS-222 (USA, Sigma). The length and weight of 100 fish were measured for each treatment. For the rice field group and pond group, respectively, the lengths were (13.56±0.49) cm and (14.10±0.23) cm, and the weights were (73.19±7.02) g and (101.20±4.57) g. The muscle quality of 30 fish from each treatment was measured, including basic nutritional components, texture characteristics, and amino acid and fatty acid compositions. The nutritional level was compared between the pond and rice field groups using the FAO/WHO amino acid score, whole egg protein comparison, protein amino acid score (AAS), chemical score (CS), and essential amino acid index (EAAI). Moreover, no significant differences between the two culture methods were observed in relation to the total ash (P>0.05). The crude protein and crude fat contents in the rice field group were significantly lower than in the pond group (P<0.05), while moisture was significantly higher (P<0.05). The viscosity of the rice field group was significantly lower than that of the pond group (P<0.05), but the cohesion and shear force were significantly higher than those of the pond group (P<0.05). There was no significant difference in the other texture indexes (P>0.05). Essential amino acids (EAA) in both groups met the FAO/WHO standard. Among the 18 amino acids measured, the total amino acids (TAA), delicious amino acids (DAA), essential and nonessential amino acids (NEAA) were significantly lower in rice field conditions than in pond (P<0.05), while the EAA/TAA and EAA/NEAA ratios were significantly lower in the pond group (P<0.05). According to the amino acid score (AAS) and chemical score (CS), glutamic acid (Glu) was the most common amino acid in both groups, while the first and second limiting amino acids were tryptophan (Trp) and valine (Val). Among the 22 fatty acids observed, the contents of tridecanoic, pentadecanoic, palmitoleic, heptadecanoic, and docosatetraenoic acids were not significantly different between the two groups (P>0.05). In contrast, the contents of linoleic, linolenic, and arachidonic acids in the pond group were significantly higher than those in the rice field group (P<0.05), while 14 other fatty acids showed significantly lower contents in the pond group (P<0.05). The monounsaturated fatty acid (MUFA) contents in the pond group were significantly higher (P<0.05), while the EPA+DHA and n-3PUFA/n-6PUFA were significantly lower than those in the rice field group (P<0.05). Overall, the contents of four main flavor amino acids (glutamic acid, glycine, alanine, and aspartic acid) in the rice field group were significantly lower than those in the pond group (P<0.05). In conclusion, rice flower carp reared in both pond and rice field is a high-quality protein source. However, different cultural environments significantly influence the rice flower carp muscle nutritional value and quality, wherein a higher nutrient composition and amino acid score were observed in the pond environment. Concerning the fatty acids content, the muscle of rice flower carp reared in rice fields had higher EPA+DHA content and N-3/N-6 polyunsaturated fatty acid ratio, which is more suitable for people with hyperlipidemia and cardiovascular diseases. In addition, in terms of texture, the muscle of rice flower carp is chewier under the rice field rearing condition. Nevertheless, N-3PUFA shortages were observed in both culture conditions. Besides, fish in the pond group had better muscle nutrition than the rice field group. Different culture conditions can change rice flower carp's fatty acid composition and content to a certain extent, but none of the two conditions tested here could completely allay the lower N-3PUFA problem. Therefore, increasing the N-3PUFA content of rice flower carp is the key to improving its nutritional value, and pond culture conditions make this process easier to be manually controlled.

Abstract:Channa argus is a bottom-dwelling freshwater fish with high nutritional and economic value and, in China, is known as the "treasure of fish”. C. argus meat is mainly used to produce fish fillets; however, head and bone are occasionally utilized in food production. The head of C. argus is rich in mineral elements, amino acids, and unsaturated fatty acids and is commonly used in China for fish head soup. The preparation of fish head soup is particularly time-consuming and can take several hours. Therefore, a high demand exists for the research and development of instant fish head soup products. With the development of modern food colloid research technology, the formation of micro/nanoparticles (MNPs) in soups and the biological effects of the tissue units as active ingredients have attracted increasing attention. In terms of the cooking process of fish soup, in addition to the heating method and cooking time, the seasoning added during cooking is also an important factor. In particular, salt is a key determinant of soup flavor. Although the content is the same, the addition of salt affects the dissolution and migration of nutrients from the fish head during the cooking process. However, the effect of the addition time of salt on the nutritious flavor during the preparation of fish soup has not been studied clearly. Furthermore, the stability of fish soup has not been adequately evaluated. In this work, the C. argus head was chosen as the base material for the soup and the nutrient migration and particles in the C. argus head soup were studied. Water-soluble protein, total sugar, total minerals, solids, nucleotides, free amino acid content, and colloidal particle stability were chosen as indicators to evaluate the transfer of nutrients and flavor. In addition, the morphological structure and distribution of MNPs in the soup were observed using laser scanning confocal microscopy (LSCM). The results showed that the content of water-soluble proteins first increased and then decreased with the delay in salt addition time. The migration trend of total sugars was similar to that of the proteins. Meanwhile, the ash content in the soup generally increased and then decreased. The change in the content of soluble solids could represent changes in various nutrients. The migration of solids and the content of ash peaked when the salting time was at 90 min. The boiled samples with salt added at different times showed significant differences in the migration of nucleotides. It could be concluded that salting was ideal at 60 min or 90 min, based on the changes in the total amount of nucleotides. Furthermore, the contents of total free amino acids and umami-flavored amino acids were also the highest in the samples with salt added at 60 min or 90 min. When the addition time was 60 min, the stability of the MNP system in the fish head soup could effectively be improved by inhibiting the aggregation of macromolecules, which would promote the retention of various nutrients. The particle size and polydispersity (PDI) were lowest in the samples with salt added at 60 min. In addition, the zeta potential value of the sample with salt added at 60 min was higher than that at 90 min. Therefore, the system was relatively stable at 60 min, which was conducive to nutrient retention. This is consistent with the results of the microstructure analysis. The maximum dissolution of nutrients and flavor substances occurred when salt was added after boiling for 60 min. Meanwhile, MNPs in the soup were stable, uniform, double-layer, spherical particles. The results of LSCM showed that nonpolar triglycerides self-assembled into spherical particles, which were located in the center of the particles, and protein molecules dispersed around or were bound to the surface of the particles. The fish head soup system was the most stable under the conditions of particle size, potential, and polydispersion index. Therefore, adding salt after boiling for 60 min was the optimal procedure for making fish soup with high nutritional value, delicious taste, and stable content. This study provides a baseline procedure for C. argus head soup with high and stable nutritional content. The results provide technical support for preparing a convenient and delicious fish head soup. People can enjoy delicious food shortly after a hard day´s work. In addition, the results showed that the timing of adding salt had a significant impact on the nutrition of fish head soup, which suggests that attention should be paid to the timing of adding salt when preparing fish head soup at home. C. argus head, a by-product of processing, can increase in value and provide a solution for producers to decrease waste and increase profits.

Abstract:Procambarus clarkii Girard is an economically important cultured shrimp in China, and lipids are an important component of its raw feed materials. Studies have shown that adequate lipid content in feed can promote the health of aquatic animals, improve the utilization efficiency of feed protein, save protein, reduce feed costs, and reduce nitrogen emissions. Insufficient lipid content in feed leads to metabolic disorders in breeding animals, reduces the utilization rate of feed protein, and is accompanied by a deficiency of fat-soluble vitamins and essential fatty acids. However, a high fat content inhibits feeding and growth and leads to excessive fat deposition in the fish body, which decreases disease resistance and is not conducive to feed processing and storage. At present, studies on the dietary lipid content of P. clarkii have mainly focused on growth performance, biochemical indices, and conventional nutritional components of muscle; however, studies on muscle quality, amino acid composition, and content have not yet been reported. This study investigated the growth performance, muscle texture indices, nutritional components, and food flavor of P. clarkii by determining the effect of dietary lipid content on the growth performance and muscle quality of juveniles. The aim of this study was to provide a reference for the development of compound feed for juvenile P. clarkii and to provide a theoretical and scientific basis for rationally evaluating the effects of fat on the muscle quality of the species. Five experimental diets with lipid levels of 2.86%, 5.11%, 7.67%, 10.19%, and 13.02% were prepared using fishmeal, soybean meal, and rapeseed meal as the main protein sources, and a mixture of fish oil and soybean oil in a ratio of 1:1 as the lipid source. A total of 600 juvenile P. clarkii with an initial body weight of (4.00±1.00) g was randomly divided into five groups (L1, L2, L3, L4, and L5 groups) with three replicates per group and 40 shrimps per replicate. They were fed five groups of experimental diets at 6:00 and 19:30 each day and cultured for 60 days. The experiment was carried out in an aquaculture pond (square, 1.0 m × 1.0 m × 0.6 m) at the Guangxi University. The results showed that as the dietary lipid level increased, the specific growth rate (SGR) and survival rate (SR) of juvenile P. clarkii first increased and then decreased and were the highest when the dietary lipid level was 7.67%. The feed conversion ratio (FCR) at first decreased before increasing again and that of the L3 group was significantly lower than that of the other groups (P<0.05). Using the specific growth rate as an evaluation index, through broken-line analysis, the optimal dietary lipid level of juvenile P. clarkii was estimated as 6.82%. Dietary lipid levels had no significant effect on the moisture, crude protein, and ash contents in the muscle (P>0.05). With the increase in dietary lipid levels, the cooking loss (CL) and crude lipid, polyunsaturated fatty acids (∑PUFA), and total amino acid (∑TAA) contents of the muscle showed an increasing trend. The content of eicosapentaenoic acid (EPA) in the L5 group was significantly higher than that in the other groups, and the docosahexaenoic acid (DHA) content in the L5 group was significantly higher than that in the L1 and L2 groups (P<0.05). With increasing dietary lipid levels, muscle hardness, elasticity, cohesiveness, and gumminess decreased and there were no significant differences between the L1 and L3 groups (P>0.05), whereas dietary lipid levels showed no significant difference in muscle saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) contents (P>0.05). The contents of umami amino acids (∑FAA), alanine (Ala), and aspartate (Asp) in the L5 group were significantly lower than those in the L1 group (P < 0.05). Under the conditions of this study, within a suitable range of fat content, growth performance increased with increased fat content; however, the addition of excessive fat cannot promote growth. According to the broken-line regression analysis of the specific growth rate, the optimal dietary lipid level was 6.82%. When the dietary lipid level was higher than 10.19%, the deposition and efficiency of muscle fat, muscle fat content, and nutrient loss increased, and the muscle texture index and Asp and Ala contents significantly decreased. The nutritional value, taste, delicious degree, and flavor of the muscle of P. clarkii were significantly decreased. As it does not affect the muscle quality of the shrimp, the dietary lipid content should not exceed 10.19%. Based on the analysis of various factors, the optimal dietary lipid content is beneficial to the healthy growth of juvenile P. clarkii and improves nutritional value and muscle quality. It is suggested that the dietary lipid supplemental level of juvenile shrimp should be 6.82%~10.19%. This study provides a theoretical basis for the scientific optimization of feed preparation for P. clarkii juvenile.