Abstract:

Abstract:

Abstract:Seagrass is a group of flowering plants capable of completing their life cycle in a marine environment. It not only provides a refuge for biodiversity and essential fish spawning and nursery grounds, but also provides ecologically and socioeconomically important services for urban coasts. Seagrass is widely distributed along temperate and tropical coastlines globally. The biodiversity corresponds with differences in the relative importance of sexual (seed production) and asexual (clonal growth) life history strategies in the maintenance of seagrass populations. Sexual reproduction in predominantly clonal marine plants increases recombination favoring adaptation and enhancing species resilience to environmental change. Flowering induction is an important link in the transition from vegetative growth to reproductive growth of seagrasses. Recent studies on seagrasses suggest that flowering intensity and frequency are correlated with global climate change, and the response of seagrasses will be more complex, and potentially more resilient than previously imagined. Seagrass environments are characterized by physical conditions, such as temperature, salinity, currents, waves, turbulence, and light. Each of these parameters has the potential to affect vegetation from the smallest (molecular and physiological) to the largest (ecosystem as well as global) scale. Based on bibliometric methods, this study summarized the development trend of seagrass flowering formation research. The SCIE database of the Web of Science core collection of the Institute for Scientific Information (ISI) was chosen as the retrieval source, and select published international journals were chosen as the research object. With "seagrass flowering" as the search keyword, a total of 300 related publications were retrieved. In terms of the number of relevant publications each year, there was an overall increasing trend, indicating that this topic has gradually received more attention in recent years, which has great research potential. This review summarizes the research progress of flowering inducement in the sexual reproduction of seagrasses and discusses the influence of physical factors on the flowering induction of seagrasses. It was previously thought that the flowering pathway initiated by seagrasses in response to environmental factors and endogenous signals may focus on the photoperiod, vernalization, and spontaneous pathways. However, the sexual reproduction of different species is different, and studying the interaction between seagrasses and their physical environment may improve understanding of the processes that influence their biology. This review focuses on the following aspects: 1) Clarifying the period of concentrated flowering of the seagrass bed, which is conducive to studying the inducement of flowering of seagrass and protecting the seagrass seed bank. 2) Discussing whether more genotypes of seagrass should be introduced for planting in the process of seagrass bed repair to avoid large-scale clonal reproduction of a single genotype of seagrass. 3) How sexual reproduction of seagrasses under adverse environments can be used to indicate changes in the climate and environment. However, existing studies have paid little attention to the sexual reproduction of seagrass beds, and do not consider the sexual reproduction rate as a long-term monitoring indicator. It is suggested that the sexual reproduction rate should be combined with environmental factors in the future long-term monitoring and assessment of the health status of seagrass beds to jointly assess their pressure status.

Abstract:Seagrass beds are a typical coastal ecosystem. To understand the temporal and spatial variation characteristics of the plankton community structure in a Zostera marina seagrass bed and an adjacent area of bare sand in Swan Lake, Rongcheng City, Shandong Province, an investigation of plankton diversity and abundance, ecological characteristics of the seagrass bed, and key environmental factors in the Z. marina seagrass bed and its adjacent bare sand area was conducted in February, May, August, and November in 2019. Canonical correspondence analysis (CCA) and redundancy analysis (RDA) were used to explore the influence of environmental factors on the diversity of plankton species. The results showed that there were 38 species of phytoplankton, belonging to 25 genera and three phyla, among which diatom species were the most abundant (89.4%), followed by dinoflagellates (7.8%). A total of 18 species of zooplankton and three species of larvae were identified (mainly crustaceans: 71.4%), and the number of plankton species was the highest in November. The annual average abundance of phytoplankton and zooplankton in the seagrass bed was 5.4×104 cells/m3 and 1.6×104 ind./m3, respectively, which were 1.4 times and 1.5 times higher than those in the bare sand area. The CCA and RDA analyses showed that the dominant plankton species in the seagrass bed were significantly correlated with water temperature, plant density, and biomass of seagrass beds, while the dominant plankton species in the bare sand area were mainly correlated with environmental factors such as water temperature, pH value, and ammonia nitrogen content. The results showed that the seagrass bed in Swan Lake supported a higher abundance and diversity of plankton compared with the bare sand area. This study provides baseline data for further elucidating the structure and function of the seagrass bed ecosystem.

Abstract:The effects of gibberellin and weak acid on the germination and physiological characteristics of eelgrass (Zostera marina) seeds were studied under laboratory conditions. The cumulative seed germination and seedling establishment rates were calculated. The dynamic changes in seed dry weight, water content, respiration rate, soluble sugar, starch, gibberellin, abscisic acid content, and α-amylase and β-amylase activities during seed germination were monitored. The effect of two exogenous germination-promoting treatments on seed germination was explored, and the physiological response process of the seeds to these treatments was analyzed. The results showed that the treatments effectively promoted seed germination and seedling establishment. The gibberellin treatment had the best germination-promoting effect, and the seed germination and seedling establishment rates were 1.6 times higher than those under the control. During the germination period of seeds in the two treatments, the α-amylase activity first increased and then decreased, the starch content showed a downward trend, and the soluble sugar content continued to increase. At the end of the experiment, the soluble sugar content of seeds in the gibberellin treatment attained the highest value, which was 3.3 times higher than the value before germination and was significantly higher than that in the weak acid treatment and control (P<0.05). The starch content of seeds in the weak acid treatment attained the lowest value, which was significantly lower than that in the other two treatments (P<0.05). There was no significant difference in α-amylase activity among the treatments (P>0.05). Principal component analysis showed that α-amylase activity and starch and soluble sugar contents of seeds were the key factors in seed germination. Comprehensive analysis showed that exogenous gibberellin treatment was an effective method to break seed dormancy. This effect was mainly achieved by regulating amylase activity, increasing starch decomposition rate, and increasing soluble sugar content to provide energy for seed germination. The results provide a theoretical and scientific basis for the rapid germination technology of eelgrass seeds.

Abstract:An increase in water turbidity is one of the main factors underlying seagrass meadow degradation. The effect of different water turbidity levels [2 (control), 10, 20, 30, and 40 NTU] on the survival, growth and physiology of eelgrass (Zostera marina) was studied through a 3-month indoor experiment. The results showed that the survival rate of eelgrass decreased gradually with an increase of water turbidity, and the survival rate under 10–40 NTU conditions was significantly lower than that of the control group (P<0.05). In particular, the survival rate of eelgrass exposed to 40 NTU was only 56.1% that of the control group. With an increase of water turbidity, the growth rate and productivity of eelgrass also showed a decreasing trend. In the 40 NTU treatment group, the internode elongation rate and leaf elongation rate of eelgrass reached minimum values, which decreased to 48.9% and 61.6% of that of the control group, respectively. Compared to the productivity of the control group, aboveground and underground productivity decreased by 64.6% and 78.8%, respectively. Correlation analysis showed that the increase of water turbidity mainly affected the growth and survival of eelgrass by affecting the content of nonstructural carbohydrates. The content of nonstructural carbohydrates in eelgrass decreased gradually with an increase of water turbidity, and the content of carbohydrates in plants exposed to 10~40 NTU was significantly lower than that in the control group (P<0.05). The aboveground soluble sugar content in eelgrass exposed to 10–40 NTU was 20.2%–74.7% lower than that in the control group. The results showed that a long-term increase of water turbidity led to a significant decrease in the nonstructural carbohydrates of eelgrass, which was not conducive to its growth and survival. The results provide a theoretical basis for clarifying the degradation mechanism of Z. marina seagrass meadows and selecting suitable restoration areas.

Abstract:Yellowtail kingfish, Seriola aureovittata, is a long-distance migratory oceanic species belonging to the Carangidae family of Perciformes, which has a global distribution and inhabits temperate and subtropical marine waters. S. aureovittata is large in size, has a fast growth rate, and is highly favored by international consumers owing to its excellent flesh taste, nutritional quality, and economic value. Furthermore, it is a promising candidate for the global farming industry and is particularly suitable for rapidly developed open ocean aquaculture in China. Currently, yellowtail kingfish aquaculture occurs in over 10 countries including Japan, Australia, New Zealand, South Africa, Chile, Greece, Holland, USA, Mexico, and China. In 2017, a great breakthrough in seedling production of S. aureovittata was achieved, and currently juveniles are mass-produced in China by combining the “engineering pond” and “land based indoor tanks” modes, which led to the rapid development of the Seriola fish farming industry in China. Nowadays, Seriola species are farmed in Liaoning Province, Fujian Province, and Shandong Province of China, and the combined annual farming yield is approximately 500 tons. However, we found that during seedling production of S. aureovittata, especially at the early larval growth stage, the hatching rate of eggs was variable among different spawning batches, and the survival of early larvae was low especially when the larvae reached 8~10 d post hatching. Occasionally, the high total death rate was attributed to the sudden “sinking death” of larvae, which may have been caused by stress as a result of changes in environmental factors. Therefore, it is necessary to determine the ecological and physiological effects of environmental factors, especially temperature and salinity fluctuations, on the early life stages of S. aureovittata under artificial breeding conditions. In the present study, the effects of two key environmental factors, temperature and salinity, on embryonic development and early larval growth of S. aureovittata were investigated using experimental ecology, morphological measurements, and molecular methods under laboratory conditions. The indexes including hatching rate of eggs, deformation rate of newly hatched larvae, absorption of yolk sac, IGF-1 gene expression, survival index (SAI), and point of no return (PNR) were determined. Moreover, the vitality of newly hatched larvae was tested and evaluated. The results showed that the highest hatching rates of 75%~81% were obtained under temperatures of 20~22 ℃, and the deformation rates of newly hatched larvae were lower than 6.7%. In addition, according to the Q10 calculation, the most appropriate water temperature range for embryonic development of S. aureovittata was confirmed to be 20~22 ℃. Meanwhile, the total length and yolk sac volume of newly hatched larvae of yellowtail kingfish hatched from the 20℃ and 22 ℃ groups were larger than those in the other temperature groups. Regarding salinity, the fertilized eggs floated on the water surface when salinity was over 30 ‰, were suspended in the water when salinity was between 20~25, and sank to the bottom of the container when salinity was lower than 15. The optimum salinity range for embryonic development of S. aureovittata was therefore determined to be 30~35, when hatching rates were between 79%~80%, and the deformation rate of newly hatched larvae was 6%. The yolk sac absorption by newly hatched larvae was measured under four temperatures (18 ℃, 20 ℃, 22 ℃, and 24 ℃). It was found that the absorption and exhaustion speed of the yolk sac increased with temperature, and the yolk sac was exhausted at 7 d post hatching at 18 ℃, whereas the time decreased to 6 d, 5 d, and 4 d at 20 ℃, 22 ℃, and 24 ℃, respectively. The highest SAI value for newly hatched larvae was observed at a salinity of 30, whereas the lowest was observed at a salinity of 10, which was consistent with the hatching results of embryos under different salinities. The highest first feeding rate of newly hatched larvae was observed at 6 d post hatching, and the PNR appeared between 7 d and 8 d post hatching at culture temperatures ranging from 20~22 ℃. IGF-1 mRNA levels in newly hatched larvae from different temperatures and salinities were detected, and significantly higher expression levels were found at temperatures of 20~24 ℃ and salinities of 30~35. Under continuous starvation conditions, the IGF-1 mRNA in larvae significantly increased at 2 d post mouth open and decreased at 3 d and 4 d, although expression levels remained relatively high, and then continually decreased to a significantly lower level as starvation continued. Results from the present study provide basic knowledge and useful tools for the construction of standardized technological methods for optimal embryonic hatching and seedling production of S. aureovittata.

Abstract:Seriola aureovittata is a long-distance migratory oceanic species belonging to the Carangidae family; it is also known as yellowtail kingfish. It inhabits temperate and subtropical marine waters worldwide. We reached a significant breakthrough in S. aureovittata seedlings production in 2017, enabling current massively juvenile rearing in China, which became a promising candidate for the farming industry, especially for the rapidly growing open ocean aquaculture. However, sudden larvae death caused by environmental changes-related stresses during the early life stages of S. aureovittata leads to great losses. Thus, exploring the physiological mechanism of larvae responses to environmental stresses during seedlings' production under artificial breeding conditions is urgently needed. Heat shock proteins (HSPs) play an important function in the physiological regulation of stress and immune responses in vertebrates, including fish. HSP70 is a member of the HSPs family studied in dozens of fish species, with physiological roles in protein homeostasis, DNA protection, and stress tolerance enhancement, among others. To investigate the possible physiological effects of HSPs on early growth and development of S. aureovittata, we cloned and obtained the full-length cDNA sequence of the hsp70 gene. Furthermore, the structure and spatial and temporal expression patterns of hsp70 during embryonic development, larval and juvenile growth and development of S. aureovittata were determined. The results showed that the full-length cDNA sequence of the hsp70 gene contains 2 332 bp, wherein the 5'-UTR is 187 bp, the ORF is 1 920 bp, and the 3'-UTR is 225 bp in length. A 639 amino acids protein with a molecular weight of 70.1 kDa is encoded by hsp70. The hsp70 spatial expression exhibited a sex dimorphism pattern, with significantly high expression levels in the gill, heart, spleen, and ovary in females, whereas in males, the significantly high expression levels were found in the pituitary, gill, head kidney, and testis. Notably, the highest hsp70 expression levels were observed in the ovary of females and the testis of males. During the S. aureovittata embryonic development, the hsp70 mRNA was detected in fertilized eggs before cleavage, indicating that hsp70 is parentally inherited. Additionally, the hsp70 mRNA could be detected at all stages of embryonic development, wherein the lowest expression levels were observed before the low blastula stage, with a significant increase at the early gastrula stage maintained until the hatching stage. The high levels of hsp70 mRNA were detected in one-day-old larva, which decreased in four-day-old larva, followed by an increase in 15-day-old larva. Remarkably, the hsp70 mRNA level once again decreased in 20-day-old larva, maintaining an average level until 60-day-old juveniles. The results from the present study may provide insights into the origin and physiological function of hsp70 during the early life stages of S. aureovittata.

Abstract:Interleukin-1 beta (IL-1β) is the quintessential pro-inflammatory cytokine, playing important roles in immune cell proliferation, differentiation, and apoptosis. The IL-1β genes have been characterized for many fish species. Unlike mammalian genes, several species of fish possess two IL-1β genes, which may be a consequence of genome duplication in particular fish lineages. Yellowtail kingfish (Seriola aureovittata) is a pelagic marine finfish species, which is an emerging candidate for the aquaculture industry. Therefore, details encompassing the role of IL-1β in the immune response aids a development strategy for economic and efficient aquaculture. In the present study, two novel il-1β molecules were identified from S. aureovittata (designated as SaIL-1β1 and SaIL-1β2). The full-length cDNA of SaIL-1β1 was 1 292 bp with a 828 bp open reading frame, encoding a polypeptide of 275 amino acids, while the full-length cDNA of SaIL-1β2 was 1 337 bp with a 960 bp open reading frame, encoding a polypeptide of 319 amino acids. Both SaIL-1β molecules contain an IL1 domain, 12 β-sheets, and a C-terminal conserved region, which are IL-1 family signature characters. A phylogenetic analysis revealed the fish IL-1βs clustered together. SaIL-1β1 and IL-1β in Seriola dumerili initially clustered together. However, SaIL-1β2 initially clustered with IL-1β in Trachinotus ovatu. Real-time PCR showed the transcripts of SaIL-1β1 and SaIL-1β2 were present in all the tested tissues, including the head kidney, spleen, liver, gill, heart, stomach, pituitary gland, muscle, and brain. Among them, the SaIL-1β1 transcripts were predominantly in the head kidney, spleen, and liver. The expression of SaIL-1β2 mRNA was predominantly in the gill, head kidney, and spleen. The high expression of SaIL-1β1 and SaIL-1β2 mRNA in the immune related organs implies a potential role in immune regulation. LPS is a pro-inflammatory endotoxin used as a standard immune activating agent. After LPS stimulation, the two SaIL-1βs transcripts were vigorously altered in the head kidney and spleen. SaIL-1β1 transcripts were significantly increased at 6 h, 12 h, 24 h, 48 h, and 72 h post-stimulation in the head kidney (10.03, 7.15, 4.09, 2.71, and 3.03-fold of the control group results, respectively) (P<0.05). Meanwhile, SaIL-1β2 transcripts significantly increased from 6 h to 24 h post-stimulation after infection in the head kidney (11.49, 4.08, and 4.70-fold of the control group, respectively) (P<0.05), had returned to normal at 48 h, and had decreased at 72 h (to 0.29-fold of the control group) (P<0.05). In the spleen, SaIL-1β1 transcripts were sharply elevated at 6 h (to 6.59-fold of control group), gradually returned to normal at 12 h, 24 h, and 48 h (3.85, 4.09, 2.17, and 2.65-fold of control group, respectively) (P<0.05), and had dropped to basal level by 72 h. SaIL-1β2 mRNA had a similar expression pattern to SaIL-1β1. SaIL-1β2 mRNA increased from 6 h to 48 h post-stimulation after infection (7.25, 3.20, 1.59, and 1.59-fold of the control group, respectively) (P<0.05) and had returned to normal by 72 h. The activated immune signaling promoted the expression of SaIL-1β1and SaIL-1β2 in the immune response, especially in the early stage, indicating they might be a pro-inflammatory cytokine in S. aureovittata. Collectively, the conserved structure and tissue distribution of SaIL-1β1 and SaIL-1β2, together with their sensitivity to LPS stimulation suggests their involvement in the immune response, providing clues to our understanding of the role of IL-1β in S. aureovittata during immune response.

Abstract:Yellowtail kingfish (Seriola aureovittata), a pelagic marine finfish species with a worldwide distribution, is regarded as an emerging candidate for the aquaculture industry owing to its fast growth, superior flesh quality, and farming suitability in both sea cages and land-based facilities in China. The species has high economic value and is the second most produced Seriola species in the world following Japanese yellowtail Seriola quinqueradiata. Researchers worldwide have studied the role of regulatory factors neuropeptide Y (NPY) in fish feeding regulation. In recent years, there has been great progress in research on food intake in fish, however, very little attention has been paid to the endocrine regulation mechanism of food intake. Methods on strengthening the production performance of fish through appetite regulation is still a hot research topic. The control of food intake and energy metabolism in vertebrates are complex processes involving several neural pathways. Some hypothalamic signals are released by peripheral tissues that are associated with energy homeostasis or nutrient availability. Among the signaling molecules involved, NPY plays a key role. NPY is recognized as one of the most effective appetite regulators, which primarily function as a signaling factor to regulate a variety of biological processes such as food intake and glucose homeostasis. The orexigenic actions of NPY have been well investigated thoroughly over the past decades. Much evidence supports that NPY´s functional role as a regulator of energy homeostasis and appetite control is conserved across vertebrates, including teleosts. In several species, including rainbow trout, Nile tilapia, and grass carp, NPY injections increase food intake, supporting an orexigenic role. In line with this, food deprivation increased npy mRNA expression in the brain, such as seen for goldfish and Johnny carp. Moreover, refeeding normalized npy mRNA abundance following food deprivation. As S. aureovittata feeds heavily and fiercely, the breeding industries need to understand its feeding control mechanism. To make real-time adjustment to feeding strategy, it is necessary to obtain high-quality and high-yield aquatic products with the least input to maximize economic benefits. As a potent appetite stimulating factor, npy has been proven to promote feeding, but this gene has not been identified in S. aureovittata. Therefore, it is necessary to explore the variable rules of the npy gene in feeding and starvation compensation mechanism, to provide the special compound feed for breeding. To gain insight into the existence of npy in S. aureovittata, we used homologous cloning, RNA extraction and reverse-transcription to obtain the ORF sequence of npy. npy belongs to the pancreatic polypeptide (PP) family, which plays an important role in appetite regulation and energy expenditure in mammals and fish. The ORF of S. aureovittata npy is 300 nucleotides in size and encodes a 99-amino-acid precusor, with a calculated molecular mass and isoelectric point of 11.24 kDa and 5.02, respectively. The precursor protein is composed of a predicted signal peptide of 28-aa in size, 36-aa putative mature peptide, a GKR protein proteolytic site, and a 32-aa C terminus of unknown function. Bioinformatics analysis on the amino acid sequence identities and evolutionary relationships of the npy was performed. Comparison of homology of the precursor peptide sequences of npy analysis revealed that S. aureovittata npy displayed a high degree of identity with the counterparts of Seriola dumerili (99.0%), Morone saxatilis (98.0%), Micropterus salmoides (96.0%), and Scophthalmus maximus (94.9%), followed by Cynoglossus semilaevis (93.9%) and Oryzias latipes (92.9%). Phylogenetic analysis highly supported that the npy of S. aureovittata was closely related to that of S. dumerili. Furthermore, using real-time quantitative PCR, we found that the npy mRNA is widely expressed in 12 tissues, with abundant expression in the brain, followed by the pituitary and stomach. In addition, except for the intestine and gonad, npy was found to have no significant difference in all other detected tissues of both sexes. To establish the functional link between npy and feeding, the expression profiles of npy mRNA during food deprivation and refeeding were examined in S. aureovittata. We detected the 7 d, 14 d, and 21 d starvation and 7 d refeeding effect on npy mRNA levels. Results showed that fasting induced an increase of npy mRNA levels in brain, pituitary, and stomach when compared to the control groups. Interestingly, the pituitary npy transcripts significantly increased after 21 d of starvation compared with the control group. In addition, refeeding normalized npy mRNA abundance following food deprivation in the brain, pituitary, and stomach. These results indicated that npy is involved in the regulation of feeding and energy homeostasis in S. aureovittata. Collectively, we provided initial evidence for the existence of npy in S. aureovittata and suggested its involvement in the regulation of feeding, which plays an important role in the starvation compensation mechanism. In summary, we obtained the ORF sequence of npy and clarified its role as a potent orexigenic peptide in feeding regulation of S. aureovittata, which would be beneficial for specific feed for this species.

Abstract:Insulin-like growth factor binding proteins (IGFBPs) play crucial roles in regulating biological activities of insulin-like growth factors (IGFs) and growth performance in vertebrates. IGFBP is a six-member protein family (IGFBP1–6) with a high affinity for IGF. It affects the distribution, stability, and biological activity of IGF by regulating the interaction between IGF ligands and receptors. Recently, IGFBP-1 was identified as a regulator of growth, reproduction, and development in bony fish, such as juvenile Atlantic salmon, where IGFBP-1 interacts with cortisol to regulate growth. IGFBP-1 regulates cell metabolism and growth through interaction with insulin in primary hepatocytes incubated in vitro in orange-spotted grouper (Epinephelus coioides). IGFBP-2 has a wide range of tissue expression characteristics in bony fish, and IGF regulation may occur through autocrine or paracrine pathways. For example, long-term fasting induced increased igfbp-2 mRNA expression in the liver of zebrafish. In addition, IGFBP-2 can inhibit the activity of IGF ligand through its high affinity with IGF to play an inhibitory and regulatory role in zebrafish growth. igfbp-2 mRNA expression was significantly up-regulated in Carassius auratus (goldfish) liver after fasting, and quickly recovered to normal levels after feeding. This indicated that igfbp-2 mRNA expression may be related to the anabolism and catabolism of goldfish, and is regulated by metabolic factors. Yellowtail kingfish (Seriola aureovittata) of the Carangidae family has high economic value and nutrition value. It is a warm and temperate oceanic fish with long distance migration characteristics that is distributed in the middle and upper oceans globally. This species is large with a fast growth rate, and is a promising candidate for aquaculture in land-based industrial circulating water system, deep-water cages, net pen systems, and aquaculture craft, etc. Our laboratory studied the regulatory mechanism of the rapid growth of yellowtail kingfish and cloned gh, igf-1, igf-2, ghr, and other growth-related functional genes to reveal their molecular regulation in early growth and development. igfbp genes regulate the growth, development, and nutrient metabolism of fish through their interaction with growth axis. The mechanism of IGFBP and how it influences the regulation of yellowtail kingfish growth is unreported. This paper further studied the growth of yellowtail kingfish by analyzing the key growth axis factors. RNAiso Plus reagent (TaKaRa) was used to extract total RNA from tissues. RNA integrity was detected by agarose gel electrophoresis, and RNA quality was determined by a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, USA). First, cDNA strand was synthesized using a PrimeScript™ RT reagent kit with a gDNA Eraser (Perfect Real-Time) reverse transcription kit (TaKaRa). Primers were designed to clone the predicted sequence of yellowtail kingfish igfbp gene according to the NCBI GenBank database. The product was amplified by polymerase chain reaction, purified from agarose gel electrophoresis, linked with T4 Ligases, transformed into Trans1-T1 Phage Resistant Chemically Competent Cell, and positive clones were selected and tested. Quantitative real time polymerase chain reaction (qRT-PCR) was used to analyze the distribution of yellowtail kingfish tissues and the expression patterns of liver tissues under different densities of industrial culture. The lengths of igfbp-1, igfbp-2a, igfbp-2b open reading frame (ORF) domains were 741 bp, 882 bp, and 801 bp, and encoded 246 amino acids, 293 amino acids, and 269 amino acids, respectively. The conserved domain of insulin growth factor-binding protein homologues (IB) was present in the N-terminus of the three igfbps, and the conserved domain of thyroglobulin type-1 repeat (Ty-1) was present in the C-terminus. They had a wide range of tissue expression characteristics and were highly expressed in the liver. There were differences between the expression of the same gene in the same tissues of male and female fish. For example, the expression of igfbp-1 and igfbp-2b genes was significantly higher in the liver of male fish compared with female fish, while igfbp-2a expression was significantly higher in the liver of female fish compared with male fish. The tissue differential expression of genes between sexes indicates that igfbp may have sex dimorphism when it plays a physiological role. However, the specific characteristics of this difference between males and females and the possible signaling pathway are unclear. The fish in the low-density group showed the greatest growth rate and the highest expression levels of igfbp-1, igfbp-2a, igfbp-2b, igf-1, and igf-2 under industrial culture conditions. These expression levels were significantly different from those of the medium and high-density groups (P<0.05). However, there were no significant differences in the expression levels of growth and liver genes between the middle and high-density groups. igfbp-1, igfbp-2a, and igfbp-2b participated in the growth regulation of yellowtail kingfish. The expression regulation of igf-1 and igf-2 had a positive synergistic effect with growth regulation of yellowtail kingfish. The ORF regions of igfbp-1, igfbp-2a, and igfbp-2b genes of yellowtail kingfish were cloned, and the structural characteristics, tissue expression characteristics, and relationships with growth performance were analyzed under different culture densities. The content of serum IGF-1, GH, cortisol, and glucose concentration was detected in the early stage. The expression trend of igfbp-1, igfbp-2, igf-1, and igf-2 was the same as that of serum IGF-1 and GH in the liver of yellowtail kingfish rapidly growing at the low-density group, but contrary to the expression trend of cortisol content and glucose concentration. This indicated that the key factors of growth axis, cortisol, and glucose participated in the growth regulation of yellowtail kingfish at different densities. However, the specific regulatory mechanism requires further study. The results provide a theoretical basis for interpreting the molecular mechanism of the growth of yellowtail kingfish and the regulation of suitable culture densities under industrial conditions.

Abstract:Sea surface temperature (SST) and chlorophyll concentration in the North Pacific in 2019–2020 were analyzed based on the data of light net fertilization fisheries, including chlorophyll-a, sea surface height (SSH), and data regarding other environmental factors. The spatial and temporal distribution characteristics of catch per unit effort (CPUE) and its relationship with key environmental factors of Sardinops sagax fishery in the Far East were analyzed by spatial superposition map and frequency analysis, empirical cumulative distribution function, K-S test, and GAM model. The results showed that the geographical fishery center ranges from 147°–153°E and 39°–43°N and moves to the northeast from April to August, returning to the southwest from September to November. According to the frequency analysis and empirical cumulative distribution function, the optimal SST and chlorophyll concentration in the central fishing area were10.0–18.0 ℃ and 0.2–0.6 mg/m3, while the optimal sea level was 0.2–0.7 m. The K-S test showed that high CPUE was closely related to SST, chlorophyll concentration, and sea surface height, and the optimal ranges were 10.9–18.9 ℃, 0.2–0.6 mg/m3, and 0.2–0.7 m, respectively. The GAM model simulation results showed that the optimal SST of high CPUE was 11.0–17.0 ℃, the optimal chlorophyll concentration was 0.3–0.8 mg/m3, and the optimal sea surface height was 0.1–0.4 m. Overall, the results showed that the optimal SST, chlorophyll concentration, and sea surface height were 11.0–18.0 ℃, 0.2–0.6 mg/m3, and 0.2–0.7 m, respectively.

Abstract:China is a major aquaculture country, with both the world´s largest aquaculture production and area, especially oyster aquaculture which accounts for 80% of the global production. However, its ascent has had numerous negative implications, requiring the development of more environmentally friendly aquaculture methods. The ecological farming model is gradually being acknowledged and encouraged as it is the result of in-depth investigations of marine ecosystems. "Integrated Multi-trophic Aquaculture" is a well-known environmentally friendly aquaculture model. The application effects have been outstanding, as it boosts high output per unit area, improves the marine environment, and improves material utilization. Filter-feeding shellfish are raised at levels that create considerable biodeposition. Particulate matter is transferred from the upper to the lower layers of the water body. Organic matter accumulates on the seafloor in the form of biological sediments, which badly influences the substrate environment, including releasing ammonia nitrogen, increasing dissolved oxygen consumption, and altering seabed biodiversity. Previous research suggests sea cucumbers absorb large amounts of organic matter-rich sediments, reducing the nutrient load caused by coastal shellfish and fish aquaculture. Therefore, a novel sustainable farming model based on the principle of multi-trophic integrated farming could be developed by using sedimentary sea cucumbers to feed on the biological sediments produced by filter-feeding shellfish. The purpose of this study was to investigate the possibility of an oyster-sea cucumber raft integrated culture. Sea cucumbers were stocked in oyster breeding cages. A raft-style integrated oyster-sea cucumber culture was attempted to improve the breeding method. This method allows oyster biological sediments to be utilized in situ, reducing oyster breeding density and maintaining economic benefits. This comparative culture experiment of integrated oyster (Crassostrea gigas)-sea cucumber (Apostichopus japonicus) raft culture utilized Sanggou Bay and Guazichang as representative oyster farming locations. We stocked C. gigas in the odd-numbered layers and A. japonicus in the even-numbered layers of the oyster cages in polyculture. Even-numbered layers had three levels of chassis: common aquaculture plate, holeless aquaculture plate, and holeless aquaculture plate with non-knot nets. The aquaculture plate was the first variable in the experiment. The stocking density of A. japonicus in each of the even-numbered layers were separated into three levels, 1, 2, and 4 ind./plate. The second variable in the experiment was stocking density of sea cucumbers. The experiment was simultaneously conducted at both sea locations. Therefore, the experimental design consisted of a three-factor and three-level experiment with a total of 18 treatment groups. During the experiment, we examined to content of: chlorophyll a, particulate organic matter, PO43–-P, NO2–-N, NO3–-N, and NH4+-N in both sea locations. The survival rate, growth performance, and condition of C. gigas and A. japonicus were compared. There was no significant difference in the contents of chlorophyll a or particle organic matter between the two marine areas (P>0.05). There were significant differences in the four nutrient salt contents between the two locations (P<0.05). There were no significant differences in the individual oyster weight or condition between the two locations (P>0.05). Only the low-stocking density sea cucumbers grew, with individual weights over 25% higher than that of the high-density individuals. Individual sea cucumber weights and survival rates in the low density treatment groups were considerably higher than those in the high density treatment groups (P<0.05). The performance of the holeless aquaculture plate considerably exceeded the common treatment group (P<0.05). The holeless aquaculture plates with sea cucumbers at a density of 1 ind./plate achieved the highest results in this study. The chlorophyll in the sea area of Sanggou Bay remained mostly unchanged in this experiment. However, the chlorophyll in the water region of Guazichang reduced when compared with that of previous data. With the recent rapid growth in the oyster industry in Rushan City, the oyster output may have reached or possibly exceeds the area´s aquaculture capacity. We advise the oyster breeding density in the Rushan sea area to be reduced to lower the breeding risk for farmers while also promoting the breeding industry´s long-term viability. In this experiment, there was no significant difference in oyster growth across the treatment groups, indicating that the integrated oyster-sea cucumber raft culture mode can lower oyster density and reduce environmental impacts. Simultaneously, breeding high-value sea cucumbers compensates for the loss of breeding income induced by the lower oyster breeding density. When compared to bottom-seeded sea cucumbers, this raft cage mode has a higher level of safety and ease of harvest. This method can be used to replenish oyster growing zones with a high density of oysters to boost the aquaculture industry´s health and long-term development.

Abstract:The sea cucumber (Apostichopus japonicus) has high economic value and is one of the important mariculture species in northern China. With the continuous development of aquaculture industry, closed or semiclosed pond aquaculture has become the main sea cucumber culture method. The ice-melting period is a special period when the ice on the pond surface melts due to the temperature rise in early spring in northern China. During this period, the ice-melting layer gradually changes the water environment of pond aquaculture from a closed state to external exchange state, resulting in the formation of thermocline and halocline areas and dissolved oxygen stratification in pond aquaculture waters. Some investigations have shown that the thermocline of pond water caused by ice-melting will cause the deterioration of water quality at the bottom of the pond and the outbreak of pathogenic bacteria, which will endanger the health of cultured organisms. Microorganisms are an important part of the pond aquaculture ecosystem. They play an important role in the ecosystem´s material circulation and energy flow and are significant to maintaining its balance. As an important part of the pond aquaculture ecosystem, sediment bacteria not only play a great role in the material circulation and energy flow processes but correlate with the growth, digestion, immunity, and diseases of aquaculture organisms. However, external physical and chemical factors easily affect the pond aquaculture ecosystem´s bacterial structure. Seasonal changes and nutrient input can modulate the species and abundance of bacteria in the pond aquaculture environment, and indirectly affect the growth and health of aquaculture organisms. In recent years, investigations have been carried out on the bacterial community structure in ponds water and sediment. Nonetheless, only few reports comprise the structure and function of the bacterial community during the ice-melting period. Therefore, it is of great theoretical and practical significance to study the structural and functional characteristics of the sea cucumber sediment bacterial community in the pond aquaculture during the ice-melting period. Therefore, this study used the typical shore-based semi-open sea cucumber pond culture in northern China as its research object. A 16S rRNA sequencing library was constructed based on the sediment bacterial community during the icebound, melting, and ablation periods and analyzed using high-throughput sequencing technology. These sequences were used to evaluate the structural characteristics of these bacterial communities and identify the dominant environmental factors affecting them. The results showed that the abundance and diversity of sediment bacterial communities showed an overall downward trend in the ice melting period, fluctuating significantly in the early stage of ice-melting (P<0.05). Compared with the icebound period, the appearance of thermocline and halocline caused drastic changes in the environmental factors of the bottom water layer. The temperature increase provides a suitable habitat for more microorganisms, accelerating enzymatic reactions and promoting microbial metabolism, which results in the up-regulation of bacteria abundance and diversity in the early melting period. As an important environmental factor in mariculture, salinity can interfere with the metabolism of microorganisms in the water and affect its diversity in sediments. This investigation showed that the bacterial community abundance and diversity in high-salinity conditions were significantly higher than those in low salinity. The substantial changes in the environmental factors destroyed the original sediment bacterial structure during the icebound period, resulting in microbial abundance and diversity fluctuation during the ice-melting period. With the gradual disappearance of the thermocline and salt layers, the pond aquaculture´s environmental factors and bacterial structure tended to be stable. In the ice-melting period, there were significant differences in the structure of sediment bacteria. The differential bacterial communities in the icebound, melting, and ablation periods were Firmicutes, Acidobacteria, and Tenericutes, respectively. Although the relative abundance ratio of microorganisms in different stages is distinct, the first dominant bacteria belong to Proteobacteria, and the relative abundance is higher than 49.04%. The secondary dominant bacteria included Bacteroides in the icebound period, Chloroflexi and Actinobacteria in the melting period, and Planctomycetes in the ablation period. The environmental factors and bacterial community structure showed significant correlation in the sea cucumber culture pond during the ice-melting period (P<0.05). Temperature, salinity, total nitrogen, and total organic carbon were the dominant environmental factors affecting sediment bacteria. This study will provide a theoretical basis for effectively managing sea cucumber pond aquaculture.

Abstract:The cobia, Rachycentron canadum (Linnaeus, 1766), is an important aquaculture species in cage and other intensive systems. This species has many advantages such as fast growth rate, excellent meat quality, and high market value, making cobia an excellent candidate species for commercial aquaculture. However, long-term artificial breeding of cobia has reduced gene exchange and population genetic diversity. To better protect germplasm resources, molecular markers provide a powerful tool in developing the breeding industry of cobia. Microsatellites are widely distributed, large in number with high polymorphism, and have long been considered important molecular markers for genetic diversity and marker-assisted breeding. More polymorphic microsatellite markers need to be developed for cobia because the number of published polymorphic simple sequence repeat (SSR) loci is very limited. In this study, Micro Satellite (MISA) software was used to identify SSR loci based on the genome sequencing data of cobia. We analyzed the distribution, quantity, and composition characteristics of the SSR loci to develop polymorphic microsatellite markers. The identified markers were used to evaluate the genetic diversity in five cultured populations. In this study, a total of 424 827 SSR loci were identified in the genome data of cobia, among which mononucleotides, dinucleotides, and trinucleotides accounted for 50.50%, 30.23%, and 14.02% of the total SSRs, respectively. Among all the repeat units contained in the total SSRs, A/T was the predominant repeat type of the mononucleotide repeats; AT/AT and AC/GT were the dominant repeat types of dinucleotides; AAT/ATT and AGG/CCT were the dominant repeat types of trinucleotides. Repeat numbers of the SSR core sequences in the genome of cobia ranged from 4 to 275 times. The predominant repeat number of the mononucleotide SSR was ten and the predominant number of the dinucleotide and trinucleotide SSR were six and four, respectively. A total of 173 518 SSR loci had a length of ≥20 bp, accounting for 47.98% of the total number of SSRs in the genome. These results indicated that the SSR loci in the genome of cobia were of a high frequency, rich variety, and with high polymorphic potential. Unigenes obtained by the genome sequencing of cobia were used to detect and analyze the SSR loci information using MISA software. The numbers and types of SSR sequences on the single-stranded DNA of the genome were counted. SSR sites in cobia genome mainly contain mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats. The screening criteria for the polymorphic SSR loci was set as “mononucleotide repeat units with repeats at least 10 times; dinucleotides with repeats at least six times; trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides with repeats at least four times”. Subsequently, based on the information of SSR loci screening, 100 candidate loci were randomly selected to design and synthesize primers for amplification. A total of 16 DNA samples from different cultured populations were used as templates. Multiple PCR amplifications were performed to screen ideal polymorphic SSR loci and suitable PCR primers. PCR products were detected using capillary fluorescence electrophoresis. The GeneMapper 4.1 software was used to analyze the accurate sites of the amplified sequences. Based on the genotyping data of the 100 SSR loci, SSR loci with high polymorphism were selected to analyze the genetic diversity of five cultured populations of cobia. The screening analysis results revealed a total of 344 820 SSR loci were detected in the genome of cobia. Among these SSR (with 1–6 nucleotide as repeat units), the top three repeat types were mononucleotide, dinucleotide, and trinucleotide, accounting for 50.49%, 30.23% and 14.02% of the total detected SSRs, respectively. Among the repeating units included in the detected SSRs, the mononucleotide repeats were dominated by A/T type, accounting for 46.34% of the total detected SSRs; AC/GT was the dominant repeat unit type of dinucleotide, accounting for 21.81% of the total detected SSRs. The number of SSR core sequence repeats in the total detected SSRs fluctuated in the range of 4 to 275 times. The predominant number of repeats of mononucleotide SSR was ten, and the predominant number of repeats of dinucleotide SSR was six. In this study, the length of ≥12 bp was set as the standard for screening high polymorphic SSR loci. A total of 361 684 SSR loci were obtained. However, the SSR loci with fragment lengths of 12–19 bp accounted the largest number, with a total of 188 166; these loci accounted for 52.02% of the total number of SSRs. Among the selected 100 candidate loci for genotyping, a total of ten polymorphic SSR markers were obtained. These markers were used in the genetic diversity analysis of five cultured populations collected in Beihai (RC-BH), Lingshui (RC-LS), Naozhou (RC-NZ), Xuwen (RC-XW), and Sanya (RC-SY). A total of 69 alleles were detected from 145 individuals, the average observed heterozygosity (Ho) was 0.628, and the average expected heterozygosity (He) was 0.706, and the mean polymorphism information content (PIC) was 0.653. The inbreeding coefficient (Fis) of the ten loci in the five cultured populations ranged from –0.317 to 0.270. The genetic distance between the five cultured populations ranged from 0.141 to 0.464, and the genetic similarity was 0.629–0.868. The results were similar to that of previous research using published markers, indicating that the polymorphic markers screened from the genome of cobia were of high accuracy and reliability. These polymorphic SSR markers provide strong support for population genetic diversity evaluation and molecular marker-assisted breeding of cobia, and provide effective technical support for the development of the cobia aquaculture industry.

Abstract:Pseudaspius leptocephalus is the famous indigenous fish in Northeast China region, with very important economic and research value. In recent years, environmental pollution and overfishing has caused it to become increasingly scarce. We performed P. leptocephalus conservation research, including clarifying the characteristics of ovarian development and the role of the sox3 gene (SRY-related high-mobility-group-box 3) in the ovarian development of P. leptocephalus. The histological characteristics of P. leptocephalus ovarian development were investigated using the paraffin section technique. The full length of the cDNA of the sox3 gene was cloned and analyzed using Rapid Amplification of cDNA End (RACE) technology. The expression pattern of the sox3 gene in different tissues and at different gonad development stages were analyzed using real-time PCR. The results revealed the ovarian cavity was clearly visible in the elliptical gonads, indicating ovary formation occurs around 45 d. At 160 d, the nucleoli of the oocytes in the ovaries were distributed along the nucleus and the testis was preparing to form seminiferous lobules and began to enter early stage Ⅱ development. At 260 d of ovarian development, the yolk nucleus appeared in the cytoplasm of some ovary oocytes, and the ovaries entered the middle stage Ⅱ phase. Also at 260 d, the number of secondary spermatocytes in the testis increased, and remained at stage Ⅱ until 360 d (when the study ceased). The differentiation of the testis began later than that of the ovaries. This result is consistent with the gonadal development in most fish. The results indicate the cDNA of the sox3 gene in P. leptocephalus is 1 800 bp (GenBank: MT952206), and encodes 299 amino acids. Among them, the protein contains an HMG conserved domain. The alignment of the amino acid sequence and the phylogenetic tree analysis indicates the SOX3 sequence in P. leptocephalus contains highly conserved regions. These regions are highly consistent with the SOX3 sequences in Danio rerio and Carassius auratus. Furthermore, the RT-PCR demonstrated that sox3 was highly expressed in the ovary, followed by the brain and eyes. Between the sexes, the expression level of the sox3 gene only varied significantly in the gonads, with the sox3 gene expression in the ovary being significantly higher than that in the testis. In the early stage of gonadal differentiation, the expression of sox3 in the ovaries is obviously higher than that in undifferentiated gonads. The lowest expression levels were detected in the testis. In the developmental stage after gonadal differentiation, sox3 was continuously expressed throughout ovarian development, while the expression in the testis remained at a low level. Therefore, the sox3 gene might play an important role in ovary differentiation and development when compared to the testis, but its specific function and mechanism require further research.

Abstract:Molluscs do not have adaptive immune cells and corresponding antibodies in their bodies. They mainly rely on the innate immune system to protect themselves from various pathogens and foreign substances to maintain normal life activities. Pattern recognition receptors (PRRs) first sense pathogen-associated molecular patterns (PAMPs) during the innate immune response, triggering specific signaling pathways and resisting pathogenic invasion. Toll-like receptors (TLRs) are widely studied PRRs and play important roles in the innate immunity of invertebrates. The first TLR was found in Drosophila, which activated the transcription factor NF-κB signaling pathway to guide early embryonic development. Later, it was proved to play an important role in the immunity of Drosophila. A typical TLR protein includes three protein domains, the extracellular domain containing two to 45 leucine-rich repeats (LRRs), the transmembrane domain, and the intracellular region containing a TIR (Toll/interleukin-1 receptor) domain. According to their structure variation in the LRR extracellular domain, TLRs can be divided into two types, namely single cysteine cluster TLR (sccTLR) and multiple cysteine cluster TLR (mccTLR). The razor clam Sinonovacula constricta is an economically important bivalve species and one of the four traditional mariculture mollusks in China. However, deterioration of the rearing environment and various bacterial and viral disease outbreaks have caused significant economic losses to the S. constricta industry. Therefore, a deep understanding of the immune defense mechanisms in S. constricta would help implement effective disease resistance strategies. In this study, we identified all TLR genes in S. constricta using whole genomic resources. Firstly, we identified the S. constricta proteins with both TIR domain (PF01582) and LRR domain (PF13855) using the InterProScan. Secondly, we further searched the whole genome DNA sequence using the TBLASTN program and the TIR-LRR protein sequences as query to identify missed TLR genes during genome annotation. The TLR protein domains identified were analyzed with the SMART software (http://smart.embl-heidelberg.de/). The TLR proteins with incomplete domains were further corrected with the FGENESH+ program on the Softberry website (http://www.softberry.com/). Finally, a total of 42 S. constricta TLR (ScTLR) genes were identified. Among them, 33 genes encode typical TLR proteins with three domains, and the remaining nine genes encode TLR-like proteins lacking some domains. The typical S. constricta TLR proteins were classified into two types and four subtypes based on the protein domain structure characteristics. The type mccTLR includes one P-TLR and seven sPP-TLRs, while the type sccTLR includes 16 sP-TLRs and nine Ls-TLRs. Furthermore, the type and number of TLR genes were compared among ten species from four classes of mollusks, including S. constricta. The results showed that two types of TLR genes, V-type and Twin-TIR TLR, identified in other mollusks were not found in the S. constricta genome. The number of TLR genes varied dramatically between different species, wherein the owl limpet (Lottia gigantea) and the common octopus (Octopus sinensis) possessed 16 and 17 TLR genes, respectively, while the American oyster (Crassostrea virginica) owned more than 130 TLR genes. Even in the same taxonomic genus, different species had a vastly different number of TLR genes, such as the Pacific oyster (Crassostrea gigas), which belongs to the same genus as the American oyster, and possessed 83 TLR genes, obviously less than the American oyster. Evolutionarily, the anciently originated mccTLR genes in mollusks did not expand in number, while the recently originated sccTLR genes largely expanded in number. The qRT-PCR tissue-specific expression analysis showed that six TLR genes randomly selected were expressed in seven tissues, including the hemolymph, gill, hepatopancreas, gonad, foot, mantle, and siphon, being highly expressed in the hemolymph, gill, and hepatopancreas. Finally, the razor clams were infected with Vibrio parahemolyticus, and the gill and hepatopancreas tissues were collected at 12 h and 24 h post-injection (hpi) for further transcriptome analysis. The results showed that nine TLR genes were differentially expressed in the gill or hepatopancreas before and after V. parahemolyticus injection. Six genes (ctg118.25, ctg118.26, ctg356.25, ctg774.6, ctg681.6, and ctg1513.5) were differentially expressed in gill at 12 hpi or 48 hpi, in which only ctg1513.5 was down-regulated and the other five were up-regulated. Three genes (ctg467.9, ctg2496.3, and ctg903.17) were differentially expressed in the hepatopancreas at 12 hpi or 48 hpi, wherein ctg467.9 and ctg2496.3 were down-regulated, and ctg903.17 was up-regulated. In summary, our findings will pave the way for investigating the functions of TLR genes in the innate immune response to different pathogens.

Abstract:Cipangopaludina cathayensis is a snail species unique to China. Domestic research on C. cathayensis has mainly focused on aquaculture technology, especially the paddy field breeding method, where water quality plays a critical role in the cultivation of C. cathayensis. Dissolved oxygen is one of the most important factors in the aquatic environment because it impacts a series of biological activities such as the growth and development, metabolism, reproduction, and breeding of aquatic animals. When the dissolution of oxygen in water is less than 2 mg/L, the water is in a low oxygen or anoxic state. Hypoxia can slow the growth and development of aquatic animals, reduce their disease resistance and reproductive ability, and, in serious cases, can lead to their death. In recent years, research on C. cathayensis has mainly focused on the anti-tumor mechanism, immune response, nutritional value evaluation, and antibiotic resistance. However, there is currently no report on the regulation of response to hypoxia in C. cathayensis either domestically or internationally. The purpose of this study was to explore the differential expression of genes in the liver of C. cathayensis under hypoxic stress. In this study, healthy C. cathayensis without mechanical damage were cultured in a hypoxia stress group (2.5 mg/L) and a normoxia (control) group (6.9 mg/L), with 90 C. cathayensis in each group and 3 replicates. For the low oxygen stress treatment, dissolved oxygen was decreased from 6.9 mg/L to 2.5 mg/L within 1 h and maintained for 24 h. The liver tissue was taken as the experimental material in both the hypoxia stress group and normoxic group. The total RNA was extracted and an mRNA library was constructed. The liver tissue samples of C. cathayensis from both groups were sequenced and analyzed using an Illumina HiSeq-2500 technology platform, and unigenes were compared and annotated in GO, KOG, Nr, and KEGG databases. The differentially expressed genes were analyzed using DESeq. Bioinformatics analysis was performed on the function of GO and KEGG of differentially expressed genes, and the key differentially expressed genes were further validated by qPCR. Transcriptome analysis results showed that 500 584 transcripts were assembled from the original data and 23 379 unigenes were obtained by sequencing, with an average length of 686.65 bp and N50 of 1 127 bp. Among the unigenes, 26 636 were found to be homologous to genes in the Nr protein database. Additionally, 22 907 unigenes were annotated in the GO database, 13 290 in the KOG database, and at least 4 179 in the KEGG database. Compared with the control group, 176 differentially expressed genes were screened in the hypoxia stress group, among which 64 and 112 were up- and down-regulated, respectively. Further, GO functional enrichment analysis found that the differential genes were mainly enriched in chitin metabolic and glucosamine-containing compound metabolic processes in the biological process. Differential genes were also enriched in collagen trimer in the cellular component and chitin binding in the molecular function. The enrichment analysis results of the KEGG pathway mainly focused on four pathway categories, namely environmental information processing, genetic information processing, metabolism, and organismal systems. Finally, the qPCR results of six key differentially expressed genes were obtained by RT-qPCR. Among the up-regulated genes under hypoxic stress were the heat shock proteins 70B2 and beta-6, and the down-regulated genes were chitinase-like protein 4, collagen alpha-1 chain (XIV), collagen alpha-4 chain (XIV), and phosphatase-related protein type 5, which confirmed the reliability of the transcriptome sequencing results. Studies have shown that, through transcriptome sequencing, the expression information of relevant functional genes in C. cathayensis liver tissues under hypoxic stress can be obtained. Among them, the expression of heat shock protein genes were up-regulated, indicating that hypoxic stress activated the physiological activities of C. cathayensis to adapt to hypoxia and protected the body from hypoxic damage. In addition, the down-regulated expression of related genes in the metabolic pathway indicates that the growth of C. cathayensis is affected under hypoxic environments. In conclusion, the results of this study provide basic data and a theoretical basis for the in-depth study of the regulatory mechanism of C. cathayensis in response to hypoxic stress.

Abstract:Brachymystax tsinlingensis is a unique cold water fish locally distributed in China, belonging to Salmoniformes, Salmonidae and Brachymystax. It is mainly distributed in the mountain streams of the Qinling mountain range, including the Shitouhe River in the northern foothills, the Heihe River in the eastern foothills and the Taibaihe and Xushuihe rivers in the southern foothills. B. tsinlingensis has high sensitivity owing to demanding natural habitat conditions and special biological properties. In the past few decades, environmental pollution, a variety of human-caused threats, and reduced resources has caused drastic declines in the wild populations of B. tsinlingensis. In 1998, the species was listed as a second-class state-protected wild animal in the China Red Data Book of Endangered Animals. Due to environmental disruption and human impacts, wild numbers of this species have declined quickly. In China, researchers have focused on B. tsinlingensis conservation. Artificial propagation is one of the most effective methods to restore the natural populations of B. tsinlingensis. In recent years, initial breakthroughs in artificial propagation techniques have aided this species, but the fry survival rate remains relatively low. During artificial breeding experiments, we identified the light environment and substrate conditions that are important factors affecting the survival rate of fry. Fry behavioral selection of light and substrate characteristics was highly significant. The aim of this study was to identify the habitat preferences and associated behavior of B. tsinlingensis. Behavioral experiments were conducted on the progeny of B. tsinlingensis in response to the light environment and substrate color. In this study, we randomly selected healthy juveniles from the same offspring batch obtained through artificial propagation as the experimental fish. The fry total length ranged from 2.23–4.57 cm, with an average of (3.31±0.67) cm. Fry weight ranged from 0.21–0.77 g, with an average weight of (0.42±0.18) g. The experimental fish were not fed 2 h before initiating the experiment. We undertook a combination of individual tests and population tests to investigate three different behavioral selection experiments on juveniles: substrate color preference with the substrate colors of black, white, and blue; light intensity preference with the light intensity of dark (from 1 lx to 5 lx), transition area (from 5 lx to 10 lx), and illuminated area (from 10 lx to 25 lx); light color preference with the light colors of yellow, red, green, or blue. The statistical analysis of the percentage of residence time and the distribution number of experimental fish in each area, enabled analysis using a selective index for the different light intensities, different light colors, and different substrate colors. All analyses used Excel 2016 and SPSS (V 25.0) software, and the statistical values were expressed as the mean ± standard deviation. The results showed that the percentage of time the individuals resided in the black substrate area was significantly higher than that in the white or blue area (P<0.05). The percentage of the population distributed in the black substrate area was also significantly higher than that in the white or blue area (P<0.05). Therefore, the majority of the fish preferred the black substrate. The fish swam slowly through the black substrate area in the substrate color experiments. Secondly, in the phototropism experiment, there was no significant variation in the duration of each individual in the three light intensity areas (P>0.05), and the percentage of the population in the illuminated area was significantly lower than that in the dark area and the transition area (P<0.05). There was no significant difference between the dark area and the transition area. The population had a negative tendency to the illuminated area, and fish were observed clustering in the dark area and the transition area. Moreover, individuals did not differ significantly in the percent of time they resided in the four light color areas (P>0.05). However, the percentage of individuals in the green light area was significantly lower than that in other areas (P<0.05). The population had a negative tendency towards the green light, and fish displayed sudden acceleration when swimming through the green area in the light color selection experiment. Consequently, the population had a more pronounced avoidance than the individual experiments, this might be related to the mutual transmission of information when residing in clusters, and the speed of information transmission in groups encouraging individuals to avoid the adverse environment. Juvenile B. tsinlingensis preferred a black substrate, avoided green light, and their optimum illumination range was 1–10 lx. The results provide scientific guidance for environmental fry rearing and releasing of B. tsinlingensis.

Abstract:The Chinese mitten crab (Eriocheir sinensis) is an important economic crab in China. Across most of China, juvenile Chinese mitten crabs are cultured in earthen ponds. Juvenile crab cultured in rice fields is mainly restricted to the northeastern region. Rice-crab coculture is a new mode of sustainable development, which has developed over the past 30 years in China with progress in field engineering, culture density of the Chinese mitten crab, and ecological benefits. However, there are no reports on appropriate feeds in rice-crab coculture. Presently, crabs reared in an earthen pond consume a traditional diet (including soybean meal, corn, bran, and wheat) or formulated feed. Due to the numerous advantages of formulated feed (including comprehensive nutrition, stable quality, and ease of feeding) the development of practical and cheap formulated feed for Chinese mitten crab is the latest focus for promoting the sustainable development of the Chinese mitten crab industry. Although researchers have developed a series of formulated feed for Chinese mitten crab, traditional feed is more commonly used in the culture of Chinese mitten crab due to farmers feeding habits and the low price of traditional products. However, numerous studies have shown that feeding Chinese mitten crabs formulated diets has many advantages, for example: it improves the survival rate, reduces the early maturity rate, improves non-specific immunity and disease resistance of juvenile crabs, improves the quality and culture performance of crab species, and economic benefits, etc. However, research were usually conducted under indoor or pond culture conditions. The culture environment of rice fields and ponds are quite different to each other. The feed sources and environmental stress on juvenile crabs may also be quite different between the culture environments. Therefore, it is important to explore the feeding mode of juvenile crabs cultured in rice fields. However, the optimum feed and the effect of different diets on the growth and physiological metabolism of Chinese mitten crab reared in rice fields remains unclear. It is important to optimize the rice-crab breeding mode to achieve a high-quality Chinese mitten crab industry. Formulated diets and traditional diets (soybean meal, bran, corn, and wheat) were fed to juvenile E. sinensis cultured in a rice field for 152 days. Each feeding group included three replicates. This study was conducted to compare formulated diets with traditional diets using culture performance, protein metabolism, digestive enzymes, and the antioxidant and immune indexes of juvenile crabs. The results showed that: (1) the body weights of female and male crabs from the formulated diet group were higher than those in the traditional diet group, but the differences were not significant (P>0.05) (Fig.1–3); (2) in terms of culture performance, the average body weight and yield of juvenile crabs, 1-year-old precocious crabs, and the overall total yield and survival rate of juvenile crabs in the formulated diet group were slightly higher than those of the traditional diet group. The feed conversion ratio of the formulated diet group was much lower than that of the traditional diet group (P<0.05); (3) in terms of protein metabolism, the total protein content in the hemolymph of both female and male crabs fed the formulated diet was significantly higher than that in the traditional diet group (P<0.05). Conversely, the contents of urea nitrogen in the hemolymph of male crabs and the activity of glutamic oxaloacetic transaminase in the hepatopancreas of the traditional diet group were significantly higher than those of the formulated diet group (P<0.05); (4) lipase in the hepatopancreas of female and male crabs in the formulated feed group was significantly higher than that in the traditional diet group (P<0.05). Conversely, the α-amylase activity of the male crabs in the formulated diet group was significantly lower than that of the traditional diet group; (5) the total antioxidant capacity, alkaline phosphatase, and acid phosphatase activities in the hemolymph of female crabs and the acid phosphatase activity in the hepatopancreas of the formulated diet group were significantly higher than those of the traditional feed group (P<0.05). In conclusion, feeding a formulated diet can improve the culture performance, promote protein deposition and lipid absorption, and enhance the antioxidant and immune capacity of juvenile E. sinensis reared in a rice field. These factors result in improved crab quality. This study provides a basis for optimizing the culture technology of Chinese mitten crab and the development of a formulated diet for improved rice-crab coculture.

Abstract:To study the classification, identification, and discrimination between different geographical populations of Triplophysa yarkandensis and explore the related otolith morphology and fish life history, this study statistically analyzed the morphological otolith indices and fish bodies of 734 T. yarkandensis from the Yarkand River, Hotan River, and Tarim River using otolith morphology and fish ecology methods. The results showed that otoliths were small in T. yarkandensis, approximately elliptic, thicker in the middle, gradually thinning to the outer edge, and with a prominent protrusion in the center of the external surface. Otolith length was obviously larger than otolith width while the excisural notch was not obvious, wherein the rostrum was developed, the ventral otolith edge was smooth with a shallow arc, and the otolith dorsal had a crest-like ridge. No significant difference between left and right lapillus morphology was observed (P>0.05). The otolith morphological indices followed a logarithmic function with the body length and weight (R2=0.48~0.62). It reflects the ontogenetic adaptation to the environment, and migration behavior mainly affects the relationship between otolith morphology and fish body morphology. The SHAPE software was used to extract the outer otolith contour of T. yarkandensis, revealing morphological differences between T. yarkandensis populations. The parameter with the largest discriminant coefficient, i.e., the one in which the morphological difference has the greatest significant effect, was screened. Therewith, the discriminant formula was set up to calculate the discriminant accuracy. Discriminant analysis between groups using fish morphology, otolith morphometry and elliptical Fourier analysis, respectively. The discriminant accuracy of the Hotan River and Tarim River populations was 96.0%, 61.4%, and 82.2%; the Yarkand River and Hotan River was 93.0%, 79.5%, and 87.9%; the Yarkand River and Tarim River populations was 96.5%, 77.5%, and 86.8%. Environmental factors such as water temperature, spatial niche adaptation, and habitat depth were the main causes of the otolith morphological changes, also affecting the behavior characteristics of typical T. yarkandensis life history, especially fish migration. In this study, the T. yarkandensis was found to live in high altitude, low habitat temperature, and high salinity and alkaline waters, so the fish body growth and the elements deposition rate onto otoliths were low. T. yarkandensis belongs to the sub-cold water and benthic fish group, which only enters deep water during overwintering in winter. In other seasons, it swims along the edge and rests in shallow depth waters, so the otolith grows slowly and has a small size. The relationship between otolith and body growth reflected the T. yarkandensis ontogenetic adaptability to its habitat. As the T. yarkandensis residence time is short in the migration area, mineral elements in the water body cannot be rapidly deposited in a short period of time, and the accelerated body growth is not completely reflected in the otolith growth. Therefore, the short-distance migration behavior under habitat fragmentation mainly affects the correlation between otolith and fish growth in T. yarkandensis. The fish otolith morphology is highly species-specific and population-specific. T. yarkandensis otolith morphology was significantly different among the geographically different populations (P<0.05). In this study, the accuracy rate (>90.0%) was slightly higher than that of elliptic Fourier analysis (>80.0%), both of which could be used as the discrimination basis parameter. However, the traditional fish otolith morphology is easy to record, as repetitive operations are robust and less affected by the environment, especially in the contents of a carnivorous fish feeding analysis; therefore, vertebrate paleontology explore has a useful application prospect in these aspects. Moreover, it could serve as an effective tool to identify fish intraspecific differences in the case of growth restriction or bodily injury. Therefore, it is of great research value to introduce the otolith morphology into the population identification of T. yarkandensis. This study explored the T. yarkandensis morphological characteristics and compared otolith morphologies to effectively identify the geographically different population, co-relating otolith shape with T. yarkandensis growth (i.e., body length and quality) and resource management, providing theoretical support to further researches about the composition and migratory population growth. The T. yarkandensis intraspecies differences in different rivers were also compared concerning fish body morphology, otolith measurements, and elliptic Fourier analysis, providing strong evidence for traditional morphological classification. The effective utilization and cost control of incomplete fish samples was greatly favored by this study. Otolith morphology was applied for the first time in the classification and population identification of T. yarkandensis, which laid a foundation for the development and research of its microchemical features and life history strategy, presenting a reference for further identification and evolutionary classification of Triplophysa, strengthening the taxonomic foundation of aquatilia, and providing scientific basis for protecting the plateau fishery germplasm resources.

Abstract:The reproductive characteristics of some hybrids from crosses of cultivated strains with wild populations are more similar to their wild parents. These hybrids form sorus twice a year in spring and autumn, unlike the conventional cultivars, which formed sorus once a year in summer. The S. japonica seedling industry begins in August in the north of China. However, hybrids form sorus in September or later. Therefore, these hybrids cannot be used as parental stock in the cultivation of summer seedlings in the north of China, hindering the promotion and application of these hybrids with excellent traits. Unfortunately, very few studies have focused on the induction and mechanism of sorus formation in kelp. It was of great significance to explore artificial induction technology for sorus formation of kelp hybrids and ensure the timely formation of sorus when the summer seedling cultivation based on an understanding of the biological process of sorus development. At present, research on the biological processes and characteristics of hybrid kelp sorus were limited. This study investigated the hybrid variety "Yudai No. 1". Discs from the kelp sporophytes were cultured in inflatable bottles. The sorus development process was divided into five stages (SA~SE) based on the appearance and morphological changes of the sorus. Samples for each stage were collected separately. The appearance, morphology, and tissue structure changes during the formation and development of sorus were systematically observed. Changes in the physiological and biochemical characteristics at different stages were also quantitatively studied. During sorus development, the surface of the sporophyte was smooth at stage SA, frosted in stage SB, noticeably protruded at stage SC, the cuticle at the apex of the paraphysis cells broken at stage SD, and the cuticle was smooth again in stage SE. The process was accompanied by the protrusion of epidermal cells (SB), the elongation of paraphysis cell (SC), the differentiation and development of sporoblast (SC, SD), and the formation and release of zoospores (SE). The cells (paraphysis cell and sporoblast) varied significantly and were constantly elongating at all stages (P<0.05). The cells were especially elongated during the stage of zoospore formation and release (SE), zoospore cells were nearly 1-fold longer than the zoospores that were not released at stage SD. During the development of S. japonica sorus, the accumulation of nitrogen by sorus continued to increase, and there was little change after reaching the maximum level at stage SD. The formation of sorus was accompanied by the accumulation of nutrients. The protein content increased significantly in the early stages of sorus development and decreased at the stage of zoospore release. The protein content was significantly higher in the SC stage than that at stage SA (P<0.05). Subsequently, the decline began after the SC stage, indicating the development of the sorus was the main biological activity, and the metabolic level was gradually reduced. Unlike that in previous studies, we identified a significant increase in the chlorophyll content during sorus development, which probably ensured all zoospores include chloroplasts. Meanwhile, hydrogen peroxide (H2O2) and superoxide anions showed similar trends of initially increasing at the beginning of sorus formation and decreasing in the later stages. Changes in the H2O2 content were highly significant in sorus formation. There were differences in the activity of different antioxidant enzymes in the process of sorus formation, among which superoxide dismutase (SOD) activity had a general downward trend, while ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT) showed a trend of rising in the early stages and then declining in the later stages. Moreover, POD, APX, and CAT activity had the significantly lowest levels at stage SC, SD, and SE, whereas the maximum levels of POD and APX were at stage SB, and maximum CAT levels were at stage SD. However, the malondialdehyde (MDA) content did not vary significantly during the whole development process. SOD activity gradually decreased throughout development, and the H2O2 content continued to increase, suggesting kelp sorus development may require hydrogen peroxide involvement. The activities of various antioxidant enzymes changed dynamically at different stages of sporangia development, and accurately regulated the oxygen species (ROS). The ROS increase in the process of sorus development did not harm any cells and ROS participated as a signaling molecule in the molecular regulation process of sorus development. In sorus development, the activity of RuBP carboxylase (RubisCO) initially decreased at stage SB and SC and then increased. There was no significant variation in the plant malate dehydrogenase activity. This study deepened the understanding of the hybrid kelp sorus formation process, physiological, and biochemical characteristics, and provided a theoretical basis for the artificial induction of hybrid kelp sorus formation in the future.

Abstract:Aeromonas salmonicida is an important pathogen that can infect a variety of marine and freshwater fish. There are five subspecies of Aeromonas salmonicida: A. salmonicida subsp. salmonicida, A. salmonicida subsp. smithia, A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. pectinolytica. Traditionally, the detection of A. salmonicida has been based on 16S rRNA sequencing and physiological and biochemical characterization, but it is difficult to identify the subspecies using these methods. Outer membrane protein (A-layer protein, VapA), which is encoded by the vapA gene and regulated by the luxS gene, is an important secretion protein of A. salmonicida. It is involved in bacterial self-agglutination induction, macrophage phagocytosis resistance, and provides protection against chemicals such as antibiotics and disinfectants. In addition, the vapA gene is also an effective molecular marker for the identification of A. salmonicida subspecies, however, gene sequencing and phylogenetic analysis are required for subspecies determination. To establish an accurate and sensitive rapid detection of A. salmonicida subspecies, in this study we tried to establish a specific PCR method for A. salmonicida subsp. salmonicida and A. salmonicida subsp. masoucida identification. Based on genome analysis, the phoB and LOC111476736 genes were used as molecular markers for PCR amplification with specific primers designed according to the sequences in the GenBank database. The target gene was amplified using the genomic DNA of A. salmonicida subsp. salmonicida or A. salmonicida subsp. masoucida, and the method was optimized to improve the efficiency and accuracy of distinguishing these two subspecies from other pathogens in aquaculture. First, the annealing temperature, primer concentration, dNTPs concentration, Mg2+ concentration, and enzyme dosage of the PCR system were optimized to improve the sensitivity of the detection method. The results showed that the primers could amplify the phoB gene fragment of 522 bp and the LOC111476736 gene fragment of 515 bp. The optimal annealing temperature of specific primers for A. salmonicida subsp. salmonicida was 64 ℃, and the optimal volume of 10 μmol/L primers, 2 mmol/L dNTPs, 25 mmol/L MgSO4, and 1 U/µL enzyme were 1.5 µL, 2.0 µL, 1.5 µL, and 0.5 μL (25 μL reaction system), respectively. The optimum annealing temperature of specific primers for A. salmonicida subsp. masoucida was 64 ℃, and the optimum volume of 10 µmol/L primers, 2 mmol/L dNTPs, 25 mmol/L MgSO4, and 1 U/µL enzyme were 0.75 µL, 1.00 µL, 1.50 µL, and 0.50 µL (25 μL reaction system), respectively. The sensitivity of the detection method was determined using a gradient diluted A. salmonicida subsp. salmonicida ATCC33658 bacterin as the template, and the target band could not be amplified when the bacterin concentration was lower than 12.8 CFU/reaction. The detection limit of A. salmonicida subsp. salmonicida based on the phoB gene sequence established in this study was 12.8 CFU/reaction. With DNA as the template, when the concentration of the DNA template was lower than 17.6 fg/reaction, the target band could not be amplified. Thus, the detection limit of specific primers based on the phoB gene sequence for A. salmonicida subsp. salmonicida was 17.6 fg/reaction. When the gradient dilution of A. salmonicida subsp. masoucida ATCC27013 bacterin was used as the template, the target band could not be amplified when the bacterin concentration was lower than 23.8 CFU/reaction. Thus, the detection limit of the method for A. salmonicida subsp. masoucida, based on the LOC111476736 gene sequence was 23.8 CFU/reaction. With DNA of ATCC27013 as the template, when the concentration of the DNA template was lower than 27.2 fg/reaction, the target band could not be amplified. Thus, in this study, the detection limit of DNA for A. salmonicida subsp. masoucida using specific primers designed according to the LOC111476736 gene sequence was 27.2 fg/reaction. The specificity of the detection method using specific primers based on the phoB and LOC111476736 genes was also determined in this study. Aquaculture pathogens or environmental bacteria, such as Vibrio anguillarum, Photobacterium damselae, Edwardsiella piscicida, Escherichia coli, Aeromonas hydrophila, Vibrio harveyi, Aeromonas encheleia, Streptococcus parauberis, Streptococcus iniae, Streptococcus dysgalactiae, and Bacillus subtilis, were used as templates for specificity testing. No specific products were found for any of the other pathogens tested. The specific PCR products could only be amplified from the bacterins of A. salmonicida subsp. salmonicida or A. salmonicida subsp. masoucida. We also tested the application of detection methods using an experimentally infected turbot as a model. Turbot was infected with A. salmonicida subsp. salmonicida strain ASS20200608XZ11L or A. salmonicida subsp. masoucida strain ASM20160705RZ6S by intramuscular injection. All turbot died within 7 days post-infection, and the liver, spleen, and kidney of moribund fish were used as templates. The results showed that the established method could accurately detect A. salmonicida subsp. salmonicida or A. salmonicida subsp. masoucida in the turbot, without nonspecific amplification in the tissues of the healthy turbot. In conclusion, we established a specific PCR method to detect two subspecies of A. salmonicida, and these methods could be used as effective tools for investigating the epidemiology of A. salmonicida.

Abstract:Sepiella japonica is widely distributed on Zhejiang and Fujian coasts and is one of the four major fishery products in the East China Sea. Its meat is delicious, has high nutritional value, and is loved by consumers. Sepia esculenta is widely distributed on the Japanese coasts and South and East China Sea waters, with the advantages of fast growth and development, short life cycle. Hence, it is an important economic cephalopod in China's northern fisheries. With the improvement of people's living standards, there is a higher demand for food quality. Red meat contains high levels of saturated fatty acids and cholesterol, which increase the risk of cardiovascular disease and colon cancer when consumed for an extended period. Cephalopods are low-fat and high-protein aquatic products, which are easier to digest and absorb than livestock meat and are widely welcomed by consumers. The quality of cuttlefish muscle is an essential factor affecting the value of cuttlefish products. So far, many scholars have studied the nutritional composition of cuttlefish muscle, but few reports have been made on its quality differences. Therefore, this study aimed to investigate the nutritional composition and quality differences of two cuttlefish muscles and evaluate their nutritional value. For that, the conventional nutrient composition, textural characteristics, cooking loss rate, formaldehyde (FA) content, amino acid and fatty acid composition, and mineral elements of S. japonica and S. esculenta were analyzed from specimens caught in large quantities in the Zhoushan area. Ten cuttlefish of each species were collected, each as an independent sample. The average carcass length for S. japonica was (104.00±0.24) mm and the average body weight (134.00±0.11) g, while for S. esculenta specimens, the averages were (162.00±0.17) mm and (356.00±0.09) g. The fresh samples were transported to the laboratory within 30 min. The basic nutrients of cuttlefish muscle were determined by the national standard method, texture characteristics by the TPA model, FA content by HPLC, amino acids content using the amino acid autoanalyzer, fatty acid content by gas chromatography, and mineral content by microwave digestion. The results showed that the crude protein and crude fat contents of S. japonica muscles were not significantly different from those of S. esculenta (P>0.05). The moisture content was significantly lower in S. japonica than that of S. esculenta (P<0.05), and the crude ash content was significantly higher than that of S. esculenta (P<0.05). The hardness, elasticity, adhesive, masticatory, and cohesive properties of S. esculenta muscles were significantly higher than those of S. japonica (P<0.05). The FA contents of the two cuttlefish muscles were 0.56 mg/kg and 1.18 mg/kg, respectively, following the national health standards. Muscles of both cuttlefish species showed 17 hydrolyzed amino acids, and the first limiting amino acid was tryptophan. Also, for both species, the ratio of essential amino acids to total amino acids was higher than 31%, and the ratio of essential amino acids to non-essential amino acids was higher than 53%. S. esculenta was closer to the ideal protein pattern recommended by the FAO/WHO, and its essential amino acid index (EAAI) was as high as 82.99. Twenty types of fatty acids were detected in the muscles of both cuttlefishes. The total contents of C20:5n-3 (EPA) and C22:6n-3 (DHA) were higher than 40% in S. japonica, which were significantly higher than in S. esculenta (P<0.05). The muscles of both cuttlefish species were rich in many inorganic elements required by humans. The two cuttlefish muscles were rich in K, P, Mg, and Ca, with the greatest difference observed for P. The P and Zn contents of S. esculenta muscles were significantly higher than those of S. japonica (P<0.05), while the I content of S. japonica muscles was significantly higher than that of S. esculenta (P<0.05), reaching 13.6 mg/kg and 9.2 mg/kg, respectively. Macronutrients had the highest content of K and P and the trace elements, Zn and I. Overall, this study showed differences in the nutritional composition and quality of the two cuttlefish muscles, but both were high-quality and low-fat protein sources with good exploitation value. This study provides the scientific basis for utilizing the cephalopod marine resources in the East China Sea.

Abstract:Nitrofurazone is a synthetic antimicrobial drug developed by the Eaton Institute in the United States in the 1950s. Nitrofurazone can play an inhibitory or bactericidal role by interfering with the glucose metabolism process and oxidase system in bacteria. Due to its strong bactericidal ability, wide antibacterial spectrum, and low price, it was widely used in animal husbandry and aquaculture. Nitrofurazone is detected in animals because it is rapidly metabolized, with a half-life of only a few hours. Semicarbazide (a typical metabolite of nitrofurazone) is detected in food-borne products in a linear proportion to the amount of nitrofurazone added to the animal. Semicarbazide binds to animal proteins to generate stable residues and is difficult to metabolize completely. The United States, European Union, China, and other countries detect and monitor semicarbazide as a marker of nitrofurazone drugs. Nitrofurazone (and its metabolite semicarbazide) have teratogenic and carcinogenic effects on the human body. Any residues in animal-derived foods can be transmitted to humans through the food chain. Long-term intake of semicarbazide in humans will cause anemia, liver necrosis, neuritis, and damages the eyeball and DNA. Therefore, the United States, the European Union, and other countries have explicitly banned its use in the food industry. China has listed nitrofurazone as a banned drug and specified that nitrofurazone and its metabolites should not be detected in animal-derived foods. Over the years, the detection of semicarbazide has been limited by the detection methods and instruments. The Ministry of Agriculture has stated the residual limit of semicarbazide as 1.0 μg/kg and assigned a supervision and sampling inspection program. Existing studies have identified the semicarbazide detected in crustacean aquatic products combines the residue caused by nitrofurazone metabolism and other obvious sources of semicarbazide, which include: 1) the presence of endogenous sources in crustacean aquatic animals; 2) the growth environment and feed intake; and 3) aquatic product processing. Previously, semicarbazide residues were generally considered to be the result of excessive nitrofurazone drug use by farmers. In recent years, the farmers state they have not used nitrofurazone during aquaculture. However, semicarbazide has been present in seafood. In 2004, Saari et al. detected semicarbazide in Procambarus clarkii that did not consume nitrofurazone and provided the first report that crustaceans may naturally produce semicarbazide, which is causing the detection of semicarbazide in many cultured crustacean aquatic animals that have not been fed nitrofurazone drugs (represented by shrimp and crab). This research confirms the presence of endogenous semicarbazones in crustacean aquatic products. In addition, the natural living environment of crustacean aquatic animals is polluted with semicarbazide due to economic human activities. Many scientists have detected the presence of semicarbazide in the waters and sediments in various regions. Concurrently, semicarbazides also contaminate aquatic plants. Semicarbazide is a new water pollutant that exists in water bodies and plants, which is continuously enriched and enters organisms. Nitrofurazone is a commonly used antibiotic for aquaculture products and is often detected when the amino residues exceed the standard levels due to illegal addition by farmers. Studies have shown that semicarbazide is also introduced through processing aquatic products, such as sodium hypochlorite disinfection resulting in an increase in the levels of semicarbazide, by azodicarboxamide through thermal decomposition producing semicarbazide and so on. The biological toxicity of semicarbazide and the food chain transfer effect have ensured semicarbazide is now an important environmental and food pollutant. In the current aquatic trade in China, the presence of endogenous semicarbazide in crustacean aquatic products has serious impacts and interferes in the detection of nitrofurazone drugs, resulting in an inability to accurately determine semicarbazide sources. It is of great importance to thoroughly analyze and understand the main sources and formation mechanism of SEM in crustacean aquatic products to ensure the healthy development of the aquaculture industry in China. At present, there are two statements on the formation mechanism of endogenous semicarbazide: arginine is involved in the urea cycle of crustacean aquatic animals and semicarbazide is produced through the oxadine intermediate. An analysis of content changes in the main substances of the urea cycle revealed the formation of endogenous semicarbazide is closely related to the guanidinyl and amide groups of arginine, citrulline, and the amide structure of urea. Arginine is a potentially important factor in the formation of endogenous semicarbazide; secondly, SEM is derived from a single cell epidermis that produces chitin. There is a single cell epidermal layer secreting chitin between the shrimp shell and shrimp meat, and the detection level of semicarbazide in the shrimp meat close to this epidermal layer was more than three times higher than the inner shrimp meat. Therefore, the semicarbazide in shrimp meat mainly originates from the epidermal layer cells producing chitin. Two inferences on the formation mechanism of exogenous semicarbazide are: the carbamate ions in hypochlorite solution may react with ammonia or acid amide in aquatic products to generate hydrazine, and hydrazine reacts with urea and other compounds through the urea cycle to generate semicarbazide, increasing the production of semicarbazide; the azodicarbonamide added in processing is degraded to biurea at high temperatures, and biurea is then converted to semicarbazide by the hydrolysis reaction. Considering the different molecular structures between nitrofurazone and biurea, the speculation that nitrofurazone is metabolized to produce biurea can be ruled out. From existing studies, azodicarbonamide is the only biological source of biurea, so biurea can be used as the corresponding target detector of azodicarbonamide. To solve the problem that endogenous and exogenous semicarbazide cannot be distinguished in aquatic products in China, the endogenous and exogenous pathways of semicarbazide and the corresponding possible formation mechanisms are reviewed in this paper. The formation pathways of endogenous semicarbazide are speculated to help solve the formation mechanism of semicarbazide in crustacean aquatic products and provide scientific data for the standardization of semicarbazide residue limits in China.