Abstract:

Abstract:

Abstract:While sex determination in animals refers to the mechanism by which individuals develop into females or males, it presents the process by which undifferentiated gonads develop into mature testes or ovaries, with sex determination as a prerequisite. Among vertebrates, fish are at a primitive stage in the process of sex evolution, and their sex-determining mechanisms are primitive, diverse, and variable, and share the sex-determining modalities of all vertebrates. The mechanisms of sex determination in fish are complex and diverse, including genetic sex determination (GSD), environmental sex determination (ESD), or a combination of both. Among them, GSD can be subdivided into two types: chromosomal sex determination and polygenic sex determination. Chromosomal sex determination is the main sex-determining (SD) gene located on the sex chromosomes that determines the sex of the offspring; whereas polygenic sex determination is the combination of multiple genes on the genome or the cumulative effect of multiple alleles on a pair of chromosomes determining the sex of the offspring; virtually all are the result of the direct or indirect action of genes. The cobia (Rachycentron canadum) is an important seawater net-pen cultured fish species in southern China. No heteromorphic sex chromosomes exist in the chromosomal karyotype of cobia, and there are no associated sex markers to distinguish between males and females. Furthermore, the sex determination and differentiation of cobia is limited, and it is difficult to rely on the external morphology of cobia to differentiate the sexes of male and female cobia parents during the breeding process. In this study, based on the whole genome information of cobia obtained in the previous period, some reported fish sex determination and sex differentiation related genes (Amh, Cyp19a1b, Dmrt1, Gdf6a/6b, and Sox9a/9b) were identified. The genes were subjected to chromosomal localization, whole genome and gene annotation files of XX/XY-type Oryzias latipes and Oreochromis niloticus, ZZ/ZW-type Cynoglossus semilaevis and Oreochromis aureus, and polygenic sex-determined zebrafish were then downloaded from the NCBI database for inter-species covariance analysis. Subsequently, phylogenetic analyses were performed, and the amino acid sequences were compared using Protein BLAST on the NCBI database. The amino acid sequences of other Osteichthyes and higher vertebrates were downloaded separately, and the phylogenetic tree was constructed using the neighbour-joining (NJ) method; followed by real-time fluorescence quantitative PCR (qRT-PCR) to detect the distribution of the expression of these genes in the tissues of adult cobia and their expression levels in gonadal tissues of different developmental stages, with the aim of screening for sex-specific genes, which will provide research materials for further exploring the mechanism of sex determination and differentiation of cobia, as well as providing a reference for the screening of sex markers in cobia. Chromosomal localization revealed that the five genes were located on seven different chromosomes of cobia without clustering. The results of covariance analyses showed that three of the five genes in cobia were covariant with XX/XY, ZZ/ZW and polygenic sex-determining fish, of which only the covariant gene for Amh was localized on the Y chromosome of XX/XY fish, all other genes are localized to autosomes. The results of interchromosomal covariance analyses showed homology between Chr7, where Amh is located, and the Y chromosome of XX/XY type fish. Phylogenetic analyses showed that each of the five genes clustered together (Sox9a and Sox9b clustered together, Gdf6a and Gdf6b clustered together), and that the scleractinians formed a separate branch in each of the gene clusters, independent of other higher vertebrates; Amh, Dmrt1, Cyp19a1b and Gdf6a were the closest relatives to Echeneis naucrates, Gdf6b was the closest relative to Lateolabrax japonicus, and Sox9a and Sox9b were the closest relatives to Seriola dumerili. The qRT-PCR results showed that Amh and Dmrt1 were specifically expressed only in the testis of cobia, which represent the male-specific genes of cobia. Gdf6a and Gdf6b were both highly expressed in the testis and skin, which may be male-biased genes. Finally, Cyp19a1b, Sox9a and Sox9b were expressed to varying degrees in tissues such as brain, skin, and heart. In the gonads of cobia at stages Ⅲ to Ⅴ, the expression of all five genes in the testis was extremely significantly higher than that in the ovary at the same period (P<0.01), and all were significant increased followed by a decrease in gonadal maturation, except for a significant decrease in Sox9b. The above results suggest that these genes may play important roles in spermathecae development in cobia, and the results may lay a foundation for revealing the regulatory mechanism of gonadal development in cobia.

Abstract:SIRT family is a NAD+ dependent class III deacetylase family, which is involved in the modification of histone or non-histone proteins. In addition to having deacetylase, some members of the SIRT family also have ADP-ribosylase and other activities, which play an important role in the regulation of energy metabolism and oxidative stress resistance. The SIRT family exists widely in prokaryotes and eukaryotes and is mainly divided into five classes. SIRT1–SIRT3 is class Ⅰ, SIRT4 is class Ⅱ, SIRT5 is class Ⅲ, SIRT6 and SIRT7 are class Ⅳ, and class U exists only in the SIRT family from archaea to bacteria. The number and distribution of SIRT genes vary among different organisms. All members of the SIRT family have been shown to be expressed in mammalian ovaries and are widely involved in the regulation of ovarian development, including meiosis regulation, energy metabolism, mitochondrial quality control, maintenance of redox homeostasis, and hormone secretion. In this study, bioinformatics methods were used to systematically analyze the chromosome distribution, gene structure, amino acid sequence, protein motifs and conserved domains, physical and chemical properties, subcellular localization, secondary and tertiary structures, phylogenetic relationships, and protein interaction networks of zebrafish SIRT family genes and to explore their expression changes at different developmental stages of follicles. The results showed that eight SIRT genes were distributed on eight chromosomes of Zebrafish, the sequence length was different [the longest was SIRT6 (140,265 bp) and the shortest was SIRT4 (7,101bp)], and the coding regions ranged from 3 to 14. The amino acid sequence similarity of zebrafish SIRT protein was low, and the 10 most conserved motifs were predicted. The adjacent homologous genes in the same branch had nearly the same motif composition, with the number of motifs varying among branches. Among the 10 motifs, motif 2, motif 3, and motif 5 were found in all zebrafish SIRT amino acid sequences, indicating that these protein motifs are highly conserved during development. Conserved domain analysis showed that all SIRT1-7 proteins contained the Sir2 domain. Analysis of physical and chemical properties of proteins showed that SIRT1 had the highest molecular weight, encoding 710 amino acids, whereas SIRT5 had the lowest molecular weight, encoding 305 amino acids. The isoelectric points ranged from 4.88 to 9.60, and all of them were hydrophilic proteins. Except SIRT3, the rest were unstable proteins. Subcellular localization prediction showed that SIRT1, SIRT4, SIRT5, and SIRT7 were located in the cytoplasm/nucleus, SIRT3.2 in the cytoplasm, SIRT6 in the nucleus, SIRT2 in the cytoskeleton, and SIRT3 in mitochondria. Secondary structure analysis showed that the SIRT family proteins had similar secondary structure, and α-helix and random curling were the main components of the protein secondary structure. The tertiary structure prediction showed that the SIRT protein family had zinc finger and Rossmann fold structures. Phylogenetic analysis showed that the fish SIRT family could be divided into three branches. The first branch consisted of three subbranches, in which SIRT1 and SIRT2 were isolated and SIRT3 and SIRT3.2 were clustered into one branch. The second largest branch consisted of SIRT4 and SIRT5, which were clustered separately into one branch. The third branch consisted of SIRT6 and SIRT7, each of which is a separate branch. The eight SIRT proteins of Zebrafish had low homology and were distributed far in the evolutionary tree. Compared with other species, zebrafish SIRT1 is closely related to rainbow trout SIRT1, whereas other family members are closely related to goldfish and electric eel SIRT. Furthermore, four family members (SIRT1, SIRT2, SIRT3.2, and SIRT4) showed collinearity between blue killifish and zebrafish, while the other family members except SIRT6 showed collinearity between goldfish and zebrafish. In addition, zebrafish SIRT4 and SIRT5 showed collinearity with two genes of goldfish. PPI prediction showed that SIRT proteins interact with ESR1, FOXOs, SOD, HSPs, etc. Real-time fluorescence quantitative PCR analysis showed that SIRT1 and SIRT2 were mainly expressed at the midvitellogenic (MV) stage during follicular development. SIRT3 and SIRT4 were mainly expressed at the full-grown immature (FG) stage. SIRT3.2, SIRT5, SIRT6, and SIRT7 were mainly expressed at the germinal vesicle breakdown (GVBD) stage. In summary, this study used bioinformatics methods for the first time to analyze chromosome localization, gene structure, amino acid sequences, physical and chemical properties, subcellular localization prediction, phylogenetic characteristics, PPI network prediction, and follicle expression at different developmental stages of zebrafish SIRT gene family. The results showed that the gene structure and amino acid sequences of eight members of the Zebrafish SIRT family were different, but all had a Sir2 conserved domain and similar protein structure. Phylogenetic analysis suggested that there may be replication or fusion events among SIRT gene family members in different species. SIRT is expressed in zebrafish follicles at different developmental stages with different expression patterns, suggesting that the SIRT plays an important role in the regulation of follicle development, providing a reference for further functional studies as well as the study of the complex molecular regulatory network of fish follicle development.

Abstract:Yellowtail kingfish (Seriola aureovittata) is a pelagic migratory fish species with a global distribution. In recent years, interest in the aquaculture of this species has increased worldwide because of its high flesh quality and fast growth. As a large and fast-swimming pelagic fish, yellowtail kingfish has good adaptability to land-based industrial recirculating and offshore net cage modes. With the development of aquaculture technology, more attention has been paid to animal behavior research, which will promote the welfare level in aquaculture. Light, sound, temperature, density, and flow velocity are important factors affecting the welfare of farmed fish. The development, morphological structure, and regulatory mechanism of sensory organs, including visual, gustatory, olfactory, and auditory organs, are becoming increasingly important in the study of animal behavior. Orthodenticle homeobox 2 (OTX2) and eye absent 1 (EYA1) play important roles in regulating the ontogeny, differentiation, and development of visual, gustatory, olfactory, and auditory organs. In order to study the expression characteristics of otx2 and eya1 in yellowtail kingfish during early development, otx2 and eya1 were identified from brain tissue with specific kits. The lengths of otx2 and eya1 open reading frame domain were 876 and 1,962 bp encoding 291 and 653 amino acids, respectively. Among these, OTX2 consists of a homologous domain from the 42nd to 93rd amino acids and a TF-Otx domain containing 82 amino acids; the C-terminal of EYA1 is an EYA domain encoding 246 amino acids. These two genes had a wide range of tissue expression characteristics, including in eye, brain, pituitary, head kidney, heart, liver, spleen, kidney, mid-gut, and ovary tissues. The highest expression level of otx2 was obtained in eye tissues followed by brain tissues, and the values in these two tissues were significantly higher than those in other tissues (P<0.05). The highest expression level of eya1 was obtained in pituitary tissues followed by ovary tissues, and the values in these two tissues were significantly higher than those in other tissues (P<0.05). During embryonic development, expression of otx2 and eya1 could be detected at each stage, and levels were upregulated at a late stage, during which otx2 and eya1 reached peak values at hatching stage and when the embryo encircled 4/5 of the yolk sac, respectively. At the larval and juvenile stages, the expression of otx2 and eya1 could be detected, with high expression levels at the early stage, during which the expression level of otx2 was first upregulated and then downregulated; the expression level at 3–25 dph (days post-hatching) was significantly higher than those at 1 dph and 30–60 dph, with the highest level reached at 20 dph (P<0.05). The expression of eya1 showed a downward trend, with the highest level reached at 1 dph (P<0.05); the expression levels at 30–60 dph were significantly lower than those at 7–25 dph (P<0.05). This study provides a molecular basis for understanding the physiological functions of otx2 and eya1 during the development of sensory organs and the regulatory mechanisms in yellowtail kingfish.

Abstract:Conger myriaster is an economic valuable fishery resources in the seas surrounding China, Korea, and Japan. C. myriaster presents an important fishing target in Chinese fishery. However, due to intense human fishing activities, C. myriaster populations have been declining steadily. Consequently, safeguarding this species has become imperative. The luteinizing hormone (LH) gene plays an important role in gamete maturation, ovulation, and steroid hormone synthesis in fish. However, the expression of reproductive axis genes and the regulation of reproductive-related hormones, specifically in C. myriaster, are not well understood. In this study, the coding sequence (CDS) of the LH gene was cloned using PCR techniques. The LH gene structure and characteristics were bioinformatically analyzed. The CDS of the LH gene consisted of 423 bp and encoded 140 amino acids. By analyzing the physical and chemical properties of the protein, the LH protein molecular weight was 15.56 kDa. The aliphatic index was 73.71 and the theoretical pI was 5.85. Based on an instability index value of 66.49, the LH protein exhibits a state of instability. Using functional domain analysis, we found that the LH protein possessed a highly conserved GHB characteristic domain (composed of 105 amino acids at 27–132). Hydrophilicity and hydrophobicity analysis of the protein revealed that the LH protein is hydrophilic. The 16th amino acid (Cys) exhibited the strongest hydrophobicity (2.233), while the 65th amino acid (Ser) exhibited the strongest hydrophilicity (–2.333). Through the signal peptide analysis and transmembrane domain analysis of LH protein, the first 1–22 amino acids function as signal peptides without containing any transmembrane domain. According to the analysis of protein glycosylation sites, the LH protein contain 1 N-glycosylation site and 3 O-glycosylation sites. The LH protein contain 17 potential phosphorylation sites. Specifically, there are 9 Serine (Ser), 6 Threonine (Thr), and 2 Tyrosine (Tyr) phosphorylation sites. Subcellular localization analysis revealed that the LH protein was mainly localized in the nucleus. The secondary structure of the LH protein mainly consists of α-helix (13.6%), β-sheet (25%), and random coil (61.4%). Upon comparing the tertiary structure of the LH protein with that of Anguilla japonica and Homo sapiens, the tertiary structure of the LH protein was similar to that of A. japanica. However, certain differences were found compared to the tertiary structure of H. sapiens. Based on the sequence alignment and phylogenetic analysis, LH in C. myriaster exhibited a closer evolutionary relationship with A. anguilla, A. marmorata, and A. japonica. Compared with A. anguilla, A. marmorata, and A. japonica, the C. myriaster LH amino acid sequence revealed the similarities of 92.14%, 91.43% and 90.71%, respectively. In addition, the LH amino acid sequence of C. myriaster compared with H. sapiens, Mus musculus and Bos taurus revealed the similarities of 43.34%, 41.73% and 41.73%, respectively. The LH of C. myriaster exhibits high homology with other teleosts, but displays low homology with mammals, which indicates that LH has evolutionary differences during evolution. In this study, we present the successful production of recombinant C. myriaster LH protein using the pET-28a(+) expression system. The restriction site NdeⅠ/XhoⅠ was inserted at both ends of LH fragment. The 439 bp fragment of LH was amplified using RT-PCR. Subsequently, the fragment was cloned into the pEASY-T1 vector to obtain recombined plasmids, which was designated as pEASY-T1-LH. The recombinant plasmid pEASY-T1-LH and the pET-28a(+) vector were double-digested using NdeⅠ/XhoⅠ enzymes. After double-digested, the fragment was successfully cloned into the pET-28a(+) vector to obtain recombined plasmids, which was designated as pET-28a-LH. In order to investigate the biological activities and physiological significance of pET-28a-LH, we used the pET protein fusion and purification system to produce pET-28a-LH in Trans1-T1 competent cell. Recombinant plasmids (pET-28a-LH) were transferred into Rosetta (DE3) cells, which were cultured under different IPTG induction conditions (0.01, 0.1, 0.5 and 1 mmol/L) at a temperature of 16 ℃ for 16 h. LH could be efficiently expressed across various IPTG concentrations and under the aforementioned induction conditions. Consequently, the subsequent experiments were conducted using the following optimized parameters: an IPTG concentration of 0.5 mmol/L, incubation at 16 ℃ for a duration of 16 h. The precipitate (insoluble fraction) was resuspended in lysis buffer. The supernatant and precipitate were analyzed by SDS-PAGE following induction of expression. SDS-PAGE analysis revealed the presence of distinct 26.5 kDa bands for the LH protein, which were soluble and present in the clear supernatant. Western blot analysis revealed that histidine tagged LH protein reacted with anti-His antibody, LH protein yielded clear bands of 26.5 kDa were detected. The observed size is in accordance with the anticipated molecular weight expression, indicating successful expression of the fusion protein carrying 6×His tag. The protein was purified using a His-tagged nickel affinity chromatography column. This result establishes a solid foundation for future utilization of recombinant LH protein in inducing sexual maturity in cultured C. myriaster, while also providing valuable insights into unraveling the regulatory mechanisms underlying LH gene-mediated steroidogenesis in C. myriaster.

Abstract:Oreochromis niloticus (Nile Tilapia), has the advantages of rapid growth, versatile feeding habits, the absence of intermuscular thorns, and robust stress resistance. Moreover, it is abundant in essential amino acids and unsaturated fatty acids, making it highly nutritious. Since 1976, the Food and Agriculture Organization of the United Nations has recognized Tilapia as a suitable fish for global cultivation. The farming model for Nile Tilapia in China is gradually shifting from traditional flowing water systems to high-density factory farming systems. However, oxygen deficiency is a common factor that leads to fish mortality in high-density intensive farming settings. Therefore, close attention must be paid to the dissolved oxygen (DO) status of aquaculture environments. DO is essential for maintaining normal metabolic activity in aquatic organisms and significantly impacts their growth, immunity, and energy metabolism. Studies have shown that fish can cope with low-oxygen environments through a series of physiological mechanisms. Under hypoxic conditions, fish utilize stored energy reserves (carbohydrates and lipids) to maintain their activities. Owing to the low utilization rate of carbohydrates in fish, utilizing lipids to provide energy is the optimal choice for them to adapt to low-oxygen environments. Owing to the low utilization rate of carbohydrates in fish, utilizing lipids to provide energy is the optimal choice for them to adapt to low-oxygen environments. Tilapia subjected to prolonged hypoxia utilized more lipids in response to hypoxic stress, suggesting that diets supplemented with appropriate lipid levels can enhance fish survival. However, the precise underlying mechanism remains unclear. This study aimed to investigate the interactive effects of DO content and fat levels on the growth performance, hepatic antioxidant capacity, immune parameters, and liver tissue structure of Nile Tilapia. The experiment was conducted using Tilapia with an initial weight of (7.62±0.29) g as the subjects. Two DO contents: hypoxia [(2.0±0.1) mg/L, group A] and normoxia [(5.0±0.1) mg/L, group B] and five fat levels (groups 1–5: 1.57%, 4.41%, 7.61%, 10.51%, and 13.01%) were applied, including three replicates per group. The culture period was 60 days. The results showed that as fat levels increased, the weight gain rate (WGR), specific growth rate (SGR), and feed efficiency of groups A and B first increased and then decreased, reaching maximum values in groups A2 and B3. At the same fat level, WGR and SGR were significantly higher in Group B than in Group A. Hepatic enzyme activities and content of various parameters, including catalase (CAT), total antioxidant capacity (T-AOC), aspartate transaminase (AST), cytochrome c oxidase (CCO), caspase-9, heat shock protein-90, malondialdehyde (MDA), hexokinase (HK), pyruvate kinase (PK), triglycerides, and hepatic lipase, were significantly affected by the interaction between oxygen levels and fat levels, whereas there was no interaction effects on adenosine triphosphatase (ATPase) activity between the two factors. With the increase in lipid levels, the CAT activity and T-AOC in group B and T-AOC in group A first increased and then decreased; the T-AOC in group A was lower than that in group B, and the B3CAT activity was significantly higher than that in the other groups. MDA content in group B increased gradually with the increase in fat level, that in group A decreased first and then increased, and that in group A was higher than that in group B. Under the same lipid level, the CCO and ATPase activities in group B were significantly lower than those in hypoxia group A. AST activity, T-AOC, and MDA content in group A were higher than those in group B, whereas caspase9 activities and HK, PK, and HSP90 contents in group B were significantly lower. Immune indicators, including immunoglobulin M, interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta, were significantly lower in group A compared to Group B and were not affected by fat level. DO and dietary fat levels significantly affected complement C3 in the liver of Tilapia. In groups A and C3, the content first decreased and then increased with an increase in fat level and was significantly lower in groups A3 and A4 than in the other groups. With increasing fat levels, the number of hepatic lipid vacuoles gradually increased in group B, whereas the cell morphology in group A2 was normal. Groups A3, A4, and B5 showed numerous lipid vacuoles, nuclear dissolution, and displacement. In summary, low oxygen levels and excessive dietary lipid levels lead to severe liver oxidative damage, fat accumulation, dysfunction of lipid metabolism, and reduced immune capacity. Within the scope of this experiment, feeding a diet with a fat content of approximately 4.41% during low oxygen conditions promoted the growth of Nile Tilapia and alleviated hypoxic stress, whereas feeding a diet with a fat content of approximately 7.61% in normoxic environments benefited the growth of Nile Tilapia.

Abstract:White shrimp (Penaeus vannamei), are native to the Pacific coastal waters of South America. With rapid growth rates and environmental adaptability, P. vannamei was introduced to China in 1988 and has since been promoted extensively for aquaculture. In 2022, its domestic production reached 2.09 million tons, accounting for one-third of the global annual aquaculture production of shrimp. P. vannamei has become a mainstay aquaculture industry of China, playing a crucial role in the economic development of coastal areas, increasing the income of fishermen, and maintaining the stable development of the rural fisheries economy. Growth traits present the most important economic trait of P. vannamei; however, with the continuous expansion of the scale culture and the deterioration of the culture environment, viral diseases are among the main causes of economic loss. Among them, white spot syndrome virus (WSSV) outbreaks have impacted many global regions since its first appearance in 1992. Infected shrimp have reduced intake, enlarged hepatopancreas, and white spots on the body. Currently, there is no effective method to prevent and control the spread of the virus, and the breeding of new varieties with both growth and WSSV resistance is urgently required. Obtaining precise genetic parameters for economic traits is the basis for developing breeding programs, and in particular, the precise assessment of heritability and genetic correlations, is an important guide in the development of selection indices, retention, and mating programs. Genetic parameter estimation mainly uses Pedigree-based Best Linear Unbiased Prediction (pBLUP). This method estimates breeding values by constructing an A matrix and correcting for different effectors and is widely used in genetic evaluation of economic traits in aquatic animals. However, pBLUP uses the A-matrix for resistance traits that are difficult to measure directly using siblings can only use 50% of the genetic variation, thus the assessment is not accurate. The use of individual typing information to construct a genomic matrix can accurately measure the true relationship between individuals, and the calculation of breeding values is performed considering the Mendelian sampling effect, which is conducive to improving the accuracy of the assessment. This assessment plays an important role in livestock and poultry breeding such as dairy cattle; however, shrimp single-tail value is low and is costly to perform at the population level. The single step genomic best linear unbiased prediction (ssGBLUP) types only some of the individuals, and composite genealogical and genotypic information is used for the breeding value assessment, which reduces the cost of individual typing. Accordingly, this method has been widely used in aquatic animals. Genomic breeding microarrays have also become an important genotyping tool in crop and livestock breeding due to their advantages of reproducibility, high accuracy, maneuverability, and low price. To assess the prospects for the application of breeding microarrays in the selection of new composite varieties for growth and WSSV resistance in the P. vannamei, this study used 59 lines of P. vannamei, totaling 1,770 individuals, and tested for WSSV infection using an independently bred high resistance line of P. vannamei. Based on the survival time of individual resistance to WSSV within the family line, 590 individuals were uniformly selected and typed using 55K SNP liquid-phase microarrays to obtain genotypic data for certain individuals. An ssGBLUP model was established by combining phenotypic values, genealogical and genotypic data, and the heritability and genetic correlation were estimated for the length of the body, the survival time of individual and half-lethal survival rate (SS50) after infection with WSSV. The heritabilities of body length and survival time in individual P. vannamei were 0.21±0.06 and 0.22±0.16, respectively, indicating medium heritability levels. The heritability of SS50 after infection with WSSV was 0.16±0.06, indicating a low heritability level. The prediction accuracy of heritability of body length based on the H-matrix was increased by 18.12% compared with the A-matrix after five-fold cross validation, and the prediction bias was not significantly different. The heritability prediction accuracy of survival time against WSSV was not significantly different from the A matrix, and the prediction bias was large in H-matrix. Furthermore, the heritability prediction accuracy of SS50 was reduced by 29.07% from the A matrix, and the prediction bias was large in the H-matrix. Based on the two-trait animal model, the estimated genetic correlation between the body length of P. vannamei and survival time of individuals against WSSV and SS50 were 0.13±0.20 and 0.30±0.22, respectively, which were not significantly different from 0 (P>0.05). The genetic correlation between the survival time of individuals against WSSV and SS50 was 0.95±0.03, which was not significantly different from 1 (P>0.05). The study showed that the genetic assessment of the growth of P. vannamei by microarray can effectively improve the accuracy of the assessment, the evaluation of WSSV survival time traits may be affected by the selection of individuals and other factors, and the prediction accuracy was not significantly improved. Finally, the survival time traits of individual anti-WSSV was better than the SS50 trait of family anti-WSSV in terms of accuracy and prediction bias. In the case that anti-WSSV survival time is highly correlated with the SS50 trait, anti-WSSV survival time can be considered as a target trait for genomic selection. In this study, we estimated the genetic parameters of growth and WSSV resistance in P. vannamei based on the typing information of 55K SNP liquid microarray “Yellow Sea Chip No.1,” which provides a reference for the application of breeding microarrays in selecting new multi-trait composite varieties of P. vannamei.

Abstract:The Pacific white shrimp (Penaeus vannamei) is currently a global mainstream aquaculture product. In recent years, as aquaculture scale has expanded, the aquaculture environment has suffered degradation, and diseases have become increasingly prevalent, severely constraining the development of the Chinese shrimp aquaculture industry. Among these diseases, acute hepatopancreatic necrosis disease (AHPND) stands out as a major cause of catastrophic economic losses. AHPND is caused by Vibrio parahaemolyticus and carried in a 69–73 kb plasmid expressing the virulence protein PirABVp. Afflicted shrimp typically exhibit symptoms such as anorexia, empty intestines and stomachs, and enlargement and softening of the hepatopancreas. Pathological studies indicate that when the toxin encounters the hepatopancreas (the target organ), it leads to the separation and disintegration of epithelial cells, turning them into substrates for bacterial replication, ultimately compromising the function of the hepatopancreas. Currently, research on AHPND primarily focuses on prevention and treatment strategies. These include adding biological agents and plant extracts to feed to enhance immunity, introducing other organisms into the water to disrupt the growth environment of V. parahaemolyticus, and analyzing the mechanism of shrimp immunity to it through bioinformatics. However, research on the distribution and amplification characteristics of pathogens within shrimp bodies is limited. Investigating the different pathways of pathogen infection in the host and the distribution and amplification characteristics of various tissues in the host after infection forms the basis of pathological research. Such research is also crucial for the scientific formulation of resistance testing methods and the accurate acquisition of resistance trait test data in breeding work. This study used selectively bred high-resistant strains of P. vannamei (average weight 35±2 g) as research subjects. Through quantitative oral infection using RT-qPCR and other techniques, P. vannamei were infected with VpAHPND in low inoculum groups (4.76×105 CFU/tail, 1.76×105 CFU/tail) (L group) and high inoculum groups (3.84×107 CFU/tail, 1.68×107 CFU/tail) (H group) at five different time points (3, 6, 9, 24, and 48 h) across nine different tissues (gill, stomach, intestine, eyestalk, muscle, hepatopancreas, fifth pleopod, abdominal nerve, and second antennae flagellum), studying the distribution and changes of PirAVp copy numbers. The amount of pVA1-like plasmid carried by V. parahaemolyticus was determined by the gene expression of the virulence protein PirAVp, which in turn represented the distribution and change characteristics of V. parahaemolyticus. The results revealed that both experimental groups experienced peak mortality between 3–6 h, with subsequent gradual decreases, and no significant difference in mortality between the two experimental groups. In the L group, plasmid copy numbers increased before decreasing in gills, eyestalk, muscle, fifth pleopod, abdominal nerve, and second antennae flagellum while they fluctuated in the stomach, intestine, and hepatopancreas. In the H group, plasmid copy numbers fluctuated in gills, intestine, eyestalk, muscle, hepatopancreas, abdominal nerve, and second antennae flagellum, with an increase before the decrease in the stomach and fifth pleopod. Among the 45 pairs of comparisons between different times within the same tissues, 32 had a significantly higher H group than the L group, 1 pair where the H group was significantly higher than the L group, one pair where the L group was significantly higher than the H group, and 1 pair where there was no significant difference. The mean plasmid copy numbers of tissues in the L group were ranked from high to low as abdominal nerve > intestine > hepatopancreas > fifth pleopod > eyestalk > muscle > gills > second antennae flagellum> stomach, and in the H group, they were ranked as abdominal nerve > intestine > gills > stomach > muscle > hepatopancreas > eyestalk > fifth pleopod > and second antennae flagellum. The average PirAVp copy numbers in the abdominal nerve and intestine tissues of both experimental groups were higher than those in other tissues, with those in the same tissues of the H group being 1.9–4.9 times higher than those in the L group. In the correlation analysis of plasmid changes between tissues of the L and H groups, there were significant correlations (P<0.05) in plasmid changes between muscle and fifth pleopod, fifth pleopod and abdominal nerve, and gills and second antennae in the L group, while there were extremely significant correlations (P<0.01) in plasmid changes between eyestalk and gills, and eyestalk and second antennae flagellum in the L group, and an extremely significant correlation (P<0.01) in plasmid changes between muscle and second antennae flagellum in the H group. Conclusively, different inoculum levels not only affect the initial distribution of pathogens in the body but also lead to different changes in the body, indicating that the changes in pathogen distribution in the body are complex and unrelated among various tissues. Therefore, further research is needed on the immune mechanisms of P. vannamei against AHPND. In the overall experiment, PirAVp was detected in all tissues at all time points in both experimental groups, with the average detection levels of abdominal nerve and intestine being higher than those of other tissues, indicating that both the intestine and abdominal nerve are suitable tissues for Vibrio attachment and proliferation, warranting further exploration of the role of abdominal nerve after infection. Although the average PirAVp copy numbers in fifth pleopod and second antennae flagellum were not the highest, they were still detectable, making them potential new materials for pathogen detection in low-impact individual vitality.

Abstract:To explore optimal large-scale breeding conditions of artificial Manila clam (Ruditapes philippinarum) seedlings, the effects of different stocking density and diet species on the growth, survival, and attachment metamorphosis of R. philippinarum larvae were investigated. Four cultivation density gradients (5, 10, 15 and 20 ind./mL) and four groups of diets (Group A Lsochrysis galbana, Group B Chaetocerossp, Group C Lsochrysis galbana + Chaetocerossp 1:2, Group D Lsochrysis galbana + Chaetocerossp 2:1) were set up. The larval shell length and survival rates were measured on the 3rd, 9th, 18th, and 27th days. The larval settlement and metamorphosis rates were evaluated on the 27th day. The results indicated that the larval shell length of (0.968±0.002) mm in diet group A was the largest on day 3. The shell length of (0.102±0.013) mm in the medium density group (10 ind./mL) was the largest, which was significantly higher than that of other density groups (P<0.05). The survival rate of bait group A (26.67%) on the 18th day was the lowest, which was significantly lower than that of the other groups. There was no significant difference in the survival rates between group C and group D, but the survival rates of group C and group B were significantly higher than those of group A and group B. There was no significant difference in the survival rate between the density groups of 5 ind./mL and 20 ind./mL; however, they were significantly lower than those of density groups of 10 and 15 ind./mL. In terms of settlement and metamorphosis, the mixed diets groups of C and D were significantly higher than those of diet groups of A and B. The density group of 15 ind./mL had the highest settlement and metamorphosis rate (17.38%), followed by 16.65% in density group of 10 ind./mL. For larval settlement and metamorphosis rates, there was no significant difference between density groups of 15 and 10 ind./mL, but significantly higher than those in density groups of 5 and 20 ind./mL. The results showed that larvae fed at early opening with Lsochrysis galbana had the highest survival rate, and larvae fed with the mixed diets showed the highest growth and metamorphosis rates. Therefore, the culture density of larvae should be maintained between 10–15 ind./mL. In summary, artificial seedling rearing of R. philippinarum, larval density should be maintained at 10–15 ind./mL. To obtain the most suitable condition for larval growth and survival, a Lsochrysis galbana diet should be selected for the early development stage and mixed diets should be adopted for the late development stage. These results provide the necessary scientific basis for the environmental factors of R. philippinarum larval growth and development and have important guiding significance and application value for improving the seed yield per water unit and stable production of seedlings of clams.

Abstract:The Chinese mitten crab (Eriocheir sinensis) is an economically important species in China, and its reproductive performance and embryo quality are particularly important for the quality of newly hatched larvae. E. sinensis is a weak crustacean thermoregulator. From November to May every year, broodstock crabs complete mating, spawning, and early larval development in the Yangtze River Estuary. During this period, they become more sensitive to environmental changes. Water temperatures that are too low or high have direct or indirect effects on the breeding of E. sinensis. Therefore, temperature is an important environmental factor affecting the breeding of E. sinensis. To explore the effects of temperature on the reproductive performance and embryo quality of female E. sinensis, reproduction and oviposition experiments of parent crabs at different temperatures (6 ℃, 9 ℃, 12 ℃, 15 ℃, 18 ℃, and 21 ℃) were conducted. Three parallel groups were set up for each temperature, and three samples were collected for each parallel group. The reproductive performance of females at different temperatures, such as mating rate, spawning rate, spawning volume, and fertility, and the quality of the embryos produced, such as egg diameter, dry weight of a single egg, and wet weight of embryos, were analyzed through observation and calculation. The crude protein content of embryos was determined using the Kjeldahl method; the total fat content was determined using acid hydrolysis; the composition and content of fatty acids were detected using gas chromatography; the moisture content of embryos was determined by taking 2–3 mg of wet embryos from each temperature group and drying them at 70 ℃ until constant weight; the ash content was determined by burning them at 550 ℃ until a constant weight; and the triglyceride, phospholipids, and cholesterol contents were determined using an enzyme immunoassay kit. The results showed that (1) with an increase in temperature, the mating rate of parent crabs increased gradually, and the spawning rate, spawning capacity, fecundity, and reproductive index first increased and then decreased. When the temperature was 15 ℃, the spawning rate, spawning capacity, fecundity, and reproductive index of female crabs were the highest, with averages of (80±10)%, (15.38±1.81)×104 grains/only, (0.23±0.03)×104 grains/only, and (20.01±1.59)%, respectively. There were significant differences with other temperature groups (P<0.05). (2) At 18 ℃, the egg diameter of the embryos was the largest, with an average of (431.17±13.69) μm. At 15 ℃, the dry weight of a single embryo was the heaviest, with an average of (31.28±4.61) μg; however, there was no significant difference between the 15 ℃ and 18 ℃ groups (P>0.05). (3) With an increase in temperature, the ash and phospholipid contents of the embryos increased gradually, whereas the total fat content decreased gradually. Triglyceride and total cholesterol contents first increased and then decreased, whereas crude protein and water contents did not change significantly. The crude protein, triglyceride, and total cholesterol contents in embryos were the highest at 18 ℃, with an average of (20.83±1.72)%, (26.2±0.71) μmol/g, and (97.3±1.19) μmol/g, respectively. (4) Twenty-one fatty acids were detected in the E. sinensis embryos, of which saturated and monounsaturated fatty acids were the highest at 12 ℃; however, there was no significant difference with other groups (P>0.05). The total contents of C20:5n3 (EPA), C22:6n3 (DHA), C20:4n6 (ARA) and polyunsaturated fatty acids in embryos at 6 ℃ were the highest, with an average of (4.26±0.85) mg/g, (4.93±0.79) mg/g, (2.96±0.44) mg/g, and (22.74±3.22) mg/g, respectively. This study showed that temperature had a certain effect on the quality of embryos produced by Chinese mitten crab, and its reproductive performance was the best at 15 ℃. This study provides basic data for the study of the breeding biology of E. sinensis and scientific support for the conservation of fishery resources of E. sinensis in the Yangtze River estuary.

Abstract:Metamorphosis is an essential stage during the ontogenesis of several important aquaculture animals, such as Apostichopus japonicus, and it covers a wide range of extensive alterations in the morphology and ecological behavior of larvae. Caspase-8 has been shown as the foremost apoptotic promoter, involved in embryonic development and immune defense processes in various aquatic animals, including fish and shellfish. To elucidate the function of the caspase-8 gene in the larval growth of the sea cucumber A. japonicus, RACE technology was employed to obtain its full-length cDNA, followed by analyses of its sequence characteristics. The expression levels of Ajcaspase-8 were assessed in adult tissues and during various larval development periods using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Moreover, the expression localization of Ajcaspase-8 and the occurrence of cell apoptosis in metamorphosizing larvae were determined via whole embryo hybridization and TUNEL techniques, respectively. The results indicated that the full-length cDNA of the Ajcaspase-8 gene was 2,790 bp, encoding 671 amino acids that included the hydrolytic active CASc domain and conserved pentapeptide QACQG characteristic of the Caspase family. Furthermore, a death receptor domain and two subunits (P20 and P10) were present in Ajcaspase-8 deduced protein, which acted as key motifs to perform the function of an initiator caspase in the apoptosis pathway. Ajcaspase-8 was clustered into a branch with Holothuria leucospilota, occupying a closer genetic distance to Oryzias latipes, indicating the advanced evolutionary status of this molecule. The RT-qPCR results showed that the Ajcaspase-8 gene in adult sea cucumbers exhibited housekeeping expression, with peak expression in coelomocytes and minimal expression in gonads (P<0.05), indicating its involvement in immunity. During the entire developmental process of A. japonicus larvae, the expression of this gene was consistently detected, showing a peak value during the doliolarial stage and second-highest value during the juvenile stage (P<0.05), both of which were significantly higher than those of other developmental stages (P<0.05). This finding suggests that Ajcaspase-8 may play an important role in the morphological reshaping of larvae during metamorphosis. The in situ hybridization analysis showed that Ajcaspase-8 positive signals were exclusively localized in the stomach of late-stage auricularia, concentrated near the hyaline sphere of doliolaria, and greatly intensified in the intestines of juveniles. This indicated that this gene was actively expressed in the redundant and digestive tissues of larvae during metamorphosis. TUNEL visualization showed that the number of apoptotic cells increased with metamorphosis, suggesting that apoptosis was an important mechanism for maintaining the homeostasis of metamorphic larvae. Taken together, these results show that the Ajcaspase-8 gene is likely to play important roles in the metamorphosis and A. japonicus attachment, suggesting that it could initiate cell apoptosis to participate in morphological reconstruction and immune defense during the crucial process of ontogenesis in this species. This study provides fundamental data for further investigation of the regulatory mechanisms underlying metamorphosis in sea cucumbers.

Abstract:Artemia is widely distributed in inland salt lakes and coastal salt fields around the world, has high nutritional value and strong palatability, and has been used as high-quality biological bait in aquaculture for a long time. Artemia can also be used as a food ingredient. Native Americans collect large quantities of Artemia, which are dried and processed into food; Libyans regard Artemia as a high-quality protein source rich in beta carotene and riboflavin and use it to make bread for sale as a delicacy. There are also reports in China that the enzymatic hydrolysate of Artemia eggs is used as a raw material to process amino acid beverages. Artemia has gradually shown great development prospects in the food industry. The growth of Artemia is mainly influenced by factors such as salinity, temperature, pH, dissolved oxygen, and light. These factors also affect physiological functions such as osmotic pressure, oxygen consumption rate, ammonia excretion rate, and reproduction to certain extents. Among them, salinity is an important factor affecting the physiological response of Artemia. Under salinity stress, the body of Artemia maintains its normal physiological functions by regulating osmotic pressure balance. The regulation of osmotic pressure balance mainly achieves ion transport through related metabolic enzymes and transporter proteins. This reverse transport process requires a large amount of energy, which comes from sugars, lipids, and proteins in the body. Therefore, the influence of salinity on the physiological functions of Artemia leads to changes in their nutritional composition, thereby affecting their growth performance and nutritional quality. Experiments were carried out in order to study the effects of different salinities on the growth performance and nutritional value of Artemia and explore its potential as raw material to develop marine food. Artemia was cultured under four different salinities (30, 60, 90, and 120) for 14 days. The body length, basic nutritional components, amino acid composition, fatty acid composition, and mineral contents of Artemia were measured and nutritional evaluations were conducted. The results showed that the body length of Artemia significantly varied under different salinities on day 14. Artemia in the salinity 30 group had the longest body length (2.05 cm) with average and specific growth rates of 0.93 mm/d and 7.65%/d, respectively. The nutritional composition of Artemia also significantly varied under different salinities, with crude protein, crude fat, ash, amino acid, and fatty acid contents ranging from 28.94% to 47.27%, 5.89% to 13.62%, 24.63% to 34.28%, 16.05 g/100 g to 37.22 g/100 g, and 2.06 g/100g to 5.48 g/100g, respectively. Among them, the salinity 30 group Artemia had the highest crude protein (47.27%) and amino acid (37.22 g/100 g) contents. Artemia in the salinity 30 group contained eight essential amino acids, which accounted for 43.42% of total amino acids, with flavor amino acids accounting for 49.52% and the first limiting amino acid being cysteine+methionine. High glutamate, leucine, and arginine contents of Artemia were observed in the salinity 30 group, with a balanced amino acids composition and an amino acid score of 91.79, which is in line with the ideal protein model recommended by the Food and Agriculture Organization/World Health Organization. Artemia in salinity 120 group had the highest crude fat (13.62%) and fatty acid (5.48 g/100 g) contents, with 2.11 g/100 gMUFA and 1.68 g/100g PUFA, respectively. EPA content was 1.13 g/100 g. Salinity levels significantly affected the mineral contents of Artemia, with the salinity 60 group showing the highest calcium, sodium, and iron contents and the salinity 90 group having the highest zinc content. In summary, the growth performance and nutritional value of Artemia in the salinity 30 group were the best. At this salinity level, the body length of Artemia were high, the growth rate was fast, and the growth performance was good. Artemia was rich in protein with balanced composition and delicious taste. Unsaturated and polyunsaturated fatty acids accounted for over 60% and 30% of the total fatty acids, respectively. Salinity significantly affected the average body length and nutritional composition of Artemia. The salinity 30 group had the longest average body length and highest protein and amino acid contents, whereas the 120 salinity group had the highest fatty acid content. Artemia is rich in protein and has a reasonable amino acid composition, making it a high-quality source of food protein. It has high nutritional value and enormous potential in the food industry. China has abundant Artemia resources; reasonable and efficient utilization of Artemia resources, research and development of Artemia-related nutritional foods, and further extension of the Artemia industry chain have great potential.

Abstract:Bangia fuscopurpurea, with high nutritional value, is a cultivated seaweed species in China. Against the backdrop of current climate change, investigating the response of B. fuscopurpurea to different temperatures, particularly high temperatures, is significant for guiding the breeding of heat-tolerant varieties and promoting the sustainable and healthy development of the industry. This study elucidates the photosynthetic and antioxidative physiological responses of B. fuscopurpurea to different temperatures (5, 10, 15, 20, and 25 ℃) by examining parameters such as the maximum fluorescence yield of photosystemⅡ(Fv/Fm), maximum relative electron transport rate (rETRmax), malondialdehyde (MDA) content, soluble protein (SP) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, ascorbate peroxidase (APX) activity, and glutathione reductase (GR) activity. The results revealed the following: (1) Overall, Fv/Fm and rETRmax of B. fuscopurpurea exhibit a trend of initially increasing and then decreasing with temperature elevation. Throughout the cultivation period, B. fuscopurpurea maintained relatively high levels of Fv/Fm and rETRmax at 15 ℃; however, within the first three days, the Fv/Fm and rETRmax values at 20 and 25 ℃ were significantly lower compared to those of other temperature groups, but by day 5, they rose to levels comparable to those of the 15 ℃ group and significantly higher than those of the 5 and 10 ℃ group. (2) The MDA content initially decreased and then increased with temperature elevation, with the MDA content at 15 ℃ being significantly lower than that of the other groups. During the early period (6 h), the MDA content was higher at low temperatures (5 and 10 ℃). However, with prolonged treatment time (five days), the MDA content significantly increased in the high-temperature groups (20 and 25 ℃). (3) The SP content was highest at 15 and 20 ℃, with no significant difference between the high (25 ℃) and low (5 and 10 ℃) temperature groups. (4) SOD, APX, GR, and CAT activities were generally significantly higher at low and high temperatures, particularly on day 5, compared with the 15 and 20 ℃ group. With prolonged cultivation time (five days), GR activity significantly increased in the 25 and 10 ℃ groups, while CAT activity significantly increased in the 5 and 10 ℃ group. These results suggest that 15 ℃ is more suitable for the growth of B. fuscopurpurea, although it also exhibits strong adaptability to high temperatures (20 and 25 ℃). During temperature stress acclimation, antioxidative enzymes play a positive role.

Abstract:Pyropia kinositae is an ideal candidate for offshore cultivation of laver, but there is room for improvement in seedling breeding techniques. Light serves as a crucial energy source and regulatory signal for algal growth and development. However, research on the role of light quality in regulating the growth and development of laver is still scarce. This study extensively investigated the effects of different light qualities (red, green, blue, and white light) on vegetative conchocelis, the transition from vegetative conchocelis to conchosporangial phase, the formation of bipartite cells in conchosporangia, and conchospore release of P. kinositae. We also examined the chlorophyll fluorescence characteristics of conchocelis under different light qualities. The results showed that relative growth rate of conchocelis under blue light was significantly higher than that under other light qualities, indicating that blue light is more suitable for the expansion of vegetative conchocelis. Under blue light, the actual quantum yield [Y(Ⅱ)], maximum electron transport rate (rETRmax), and the minimum saturating irradiance (Ek) of conchocelis as well as the maximal quantum yield of Photosystem Ⅱ (Fv/Fm) of conchospores were significantly higher than those under other light qualities. This suggests that blue light significantly enhances the photosynthetic efficiency of conchocelis and improves its electron transfer activity and phototolerance under high light conditions. In contrast, Y(Ⅱ) and rETRmax of conchocelis under red light were significantly lower than those under other light qualities. Under blue light, the conversion rate of conchocelis to conchospores (48.7%) and the rate of bipartite cells in conchosporangia formation (26.7%) were significantly higher than those under other light qualities, indicating that blue light significantly promotes the synchronous maturation of conchocelis, followed by white and green light, whereas red light did not show a significant effect. When inducing the conchospore release from conchocelis with consistent maturity, the early release of conchospores did not significantly vary under white, red, and green light, but a significant increase was observed under blue light, indicating that blue light promotes the release of bipartite cells. The germination rate of conchospores under red and green light was low (<10%), whereas it was significantly higher (>80%) under blue light compared with the other light qualities. These results provide preliminary insights into light quality conditions and the photosynthetic physiological basis for promoting the growth and development of P. kinositae conchocelis and muturation and release of conchospores, offering theoretical support for the regulation of light in seedling production.

Abstract:Sea cucumber (Apostichopus japonicus) is an important marine aquaculture species in China. In 2022, the total area of sea cucumber aquaculture in China reached >3.7 million acres, with a breeding yield of 24.85 tons. Sea cucumber aquaculture is the largest single variety of marine aquaculture species in China. With the increase of sea cucumber aquaculture scale and density, its feeding efficiency is low, which leads to low utilization rate of feed. It is easy to cause residual bait to mold and decay, polluting water quality and leading to frequent sea cucumber diseases. Therefore, antibiotic abuse has become abundant. However, the extensive use of antibiotic drugs poses ecological and food safety risks. Therefore, developing environmentally friendly and healthy feed additives that have feeding, antibacterial, and immune enhancing effects is urgently required. Our research group screened various sea cucumber attractants in the early stage and selected allicin for further feeding experiments based on the initial experimental results. To investigate the effects of adding allicin to feed on the growth, digestion, immune performance, and gut microbiota structure of sea cucumber (Apostichopus japonicus), healthy sea cucumber with an initial body weight of (50.25±3.21) g was used as the research object. Feed supplemented with 0% (control), 0.2%, 0.4%, and 0.6% allicin was used as feed for 45 days. The growth rate, immune and digestive enzyme indicators, as well as differences in gut microbiota structure of different experimental groups were determined. Weight gain and specific growth rates of the experimental group with added allicin were significantly higher than those of the control group (P<0.05), with the weight gain rate and specific growth rate of the group with added 0.4% allicin being (29.49±2.07)% and (0.57±0.13)%/d, respectively, significantly higher than the other groups (P<0.05). As the amount of allicin added increases, the activities of digestive enzymes such as trypsin, amylase, and lipase, as well as non-specific immune enzyme activities such as alkaline phosphatase, acid phosphatase, lysozyme, and superoxide dismutase in body fluid, all showed a trend of first increasing and then decreasing. Except for the ACP index, the digestion enzyme and non-specific immune enzyme activities of the 0.4% experimental group were significantly higher than those of the other groups (P<0.05). There was no significant difference in the number of OTUs in the gut microbiota of sea cucumber. The Chao1 index showed a trend of first increasing and then decreasing with the increase of allicin addition, while diversity indices such as ACE, Shannon, and Simpson indices showed a decreasing trend with the increase of allicin addition. The diversity index of the 0.6% experimental group was the smallest. The addition of allicin affected the gut microbiota structure of sea cucumber, with an increasing trend in the abundance of Rhodobacter, a decreasing trend in the abundance of Escherichia coli, and an initial increase followed by a decrease in the abundance of Clostridium. When the allicin addition was 0.6%, the microbial community structure changed significantly (P<0.05). LEfSe analysis showed that Chitinophagales, Ruminococcacaea, Gardnerella, and Bifidobacteriales were significantly dominant bacteria in the control group (P<0.05), indicating that these bacteria were significantly inhibited with the addition of allicin. The research results indicate that adding an appropriate amount of allicin to feed can improve the growth performance of sea cucumber, promote digestive and non-specific immune enzyme activity, increase the abundance of beneficial bacteria while inhibiting the proliferation of harmful bacteria, and change the structure of sea cucumber gut microbiota. The appropriate amount of allicin added to feed was 0.4%. To our knowledge, this is the first report to demonstrate the effect of different garlic concentrations on the growth performance and intestinal microbiota of sea cucumber. Dietary garlic supplementation was intestinal microbiota for sea cucumber when administered as feed additive in terms of promoting growth and inducing changes in the intestinal microbiota. Finally, our research findings suggest that dietary garlic supplementation may represent an antibiotic as growth promoter in aquaculture.

Abstract:Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a shrimp disease that poses a substantial threat to the shrimp farming industry. Owing to the significant economic impact of IHHNV on the global shrimp farming industry, the World Organization for Animal Health (WOAH) has listed IHHNV as a notifiable crustacean pathogen. The primary IHHNV detection method usually involves capturing individual shrimp for molecular biological testing. Using environmental DNA (eDNA) technology, which allows rapid and economical pathogen monitoring directly from environmental samples, has rapidly developed for aquatic animal pathogen detection applications. eDNA is used to detect aquatic pathogens, such as external parasites in fish and crustacean diseases, including white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), and IHHNV. For instance, for a method has been developed for detecting Cyprinid herpesvirus 3 (CyHV-3) in environmental waters using virus concentration methods and TaqMan polymerase chain reaction (PCR). The concentration-PCR method achieved an average concentration recovery rate of 67.11% for IHHNV detection in environmental waters based on eDNA principles and techniques. Shrimp carrying the IHHNV pathogen can spread the disease to healthy populations, inducing epidemics. Monitoring pathogens in the water environment is a more direct and effective method compared with testing cultivated shrimp for biosecurity investigations and risk assessments. While such eDNA methods are well-studied, research regarding such applications for soil and sediment is limited. Viruses can persist in pond soil and sediments, serving as natural virus reservoirs and providing potential pathways for virus transmission. However, no studies have monitored the presence of IHHNV in natural environment sediments, which is accompanied by a lack of reliable detecting and quantifying IHHNV detection methods for environmental sediments. eDNA methods enable an effective understanding of pathogen transmission mechanisms and the timely establishment of control measures during disease outbreaks. As a promising tool, eDNA detection has significant application prospects in monitoring aquatic animal diseases. This study aimed to evaluate the effectiveness and feasibility of using eDNA technology to detect the shrimp disease pathogen, IHHNV. Different particle size substrates of pond sediments were selected as study subjects. Extraction conditions were optimized using three commercial kits to evaluate the eDNA extraction effects under different substrates and to detect the lowest nucleic acid detection limit of IHHNV using real-time fluorescent quantitative PCR (RT-qPCR). To verify nucleic acid extraction effectiveness from sediments, three different kits were applied to extract DNA from shrimp tissue in both mud and sand substrates, followed by PCR amplification. Considering factors such as kit price, extraction effect, and duration, Kit B and A were selected for nucleic acid extraction from sand and mud sediments, respectively. RT-qPCR amplification of IHHNV in two types of substrate sediments at different addition volumes were observed. As the addition volume of IHHNV-containing shrimp tissue homogenate decreased, the viral load of IHHNV decreased accordingly. The minimum detectable addition volume for IHHNV in sand sediments was 5 μL, with a viral load of 1.52×102 copies/μL; whereas the minimum detectable addition was 10 μL for mud sediments, with a viral load of 1.32×102 copies/μL. The original viral load in 5 μL and 10 μL homogenate volumes were 9.94×102 and 1.72×103 copies/μL, respectively. Compared to the original viral load added, the recovery rate in sand and mud sediments were approximately 15.30% and 7.70%, respectively. The minimum detection limit concentration of different pond sediment components varied by less than an order of magnitude, showing no significant difference in extraction effects, making it suitable for IHHNV detection in sediments. The optimized eDNA technique and RT-qPCR method for cultured shrimp tissue samples were applied to investigate the presence of IHHNV in the farming environment. IHHNV positives were detected in both sediment and shrimp farming ponds between July and September; however, the positive detection rate was lower in sediment than in shrimp (Penaeus vannamei). The study demonstrates that the detection results of pond sediments and cultured P. vannamei samples are consistent, indicating that pond sediments effectively reflect the IHHNV infection status of shrimp farms. IHHNV RT-qPCR detection in pond sediments during the cultivation period from July to September 2022 were observed. IHHNV loads reached 102 copies/μL level in all six farming ponds. Notably, the IHHNV load in the sediment of pond 6-1 reached up to 4.88×102 copies/μL. Viral loads in shrimp tissue samples reached up to 102–103 copies/μL, indicating that IHHNV loads in shrimp tissue samples were higher than those in pond sediments. This study provides a reliable technical method to evaluate IHHNV detection methods in shrimp farming pond sediments, offering a scientific basis for monitoring the health status of cultured animals and supporting the application of eDNA methods for monitoring shrimp pathogens.

Abstract:Whiteleg shrimp (Penaeus vannamei) significantly contributes to the Chinese aquatic product market. However, continuous intensification has led to eutrophication in water bodies, resulting in frequent cyanobacterial blooms. These blooms release microcystins, which pollute water and cause substantial death rates among cultured organisms, posing a serious threat to agriculture and public health. Microcystin-LR (MC-LR) is the most prevalent and highly toxic variant. MC-LR is toxic to aquatic organisms and impacts terrestrial organisms and human health in several ways. These significant effects have attracted widespread attention in the academic community. MC-LR is hepatotropic and accumulates in the hepatopancreas after entering the body. Numerous reports have described the toxicological effects of MC-LR from various perspectives; however, its potential biological processes remain highly complex and may involve multiple signaling pathways in P. vannamei. Most experiments investigating the acute exposure of shrimp to compounds use blood sinus injection or intramuscular injection. However, these methods can potentially cause tissue damage and change immune indicators. Reverse-gavage reduces tissue damage and directly affects the hepatopancreas. Furthermore, reverse-gavage can also simulate reactions to toxic substance under natural conditions. First, the MC-LR solution was mixed with red edible dye, which was then slowly dripped into the anal cavity of the experimental group using an automatic pipette. The inoculation solution entered the midgut from the hindgut and ceased when the red color reached the hepatopancreas. After 24 h, the cephalothoraxes was dissected to obtain the hepatopancreas tissue. Subsequently, transcriptome sequencing technology was used to identify the differentially expressed genes related signaling pathways and metabolic pathways in the hepatopancreas of P. vannamei under MC-LR treatment. The results of the transcriptome sequencing were validated using quantitative real-time PCR. MC-LR induced significant differential expression of 1 994 genes compared to the control group. Of these, 1 164 were up-regulated and 830 were down-regulated. The differentially expressed genes were categorized based on biological processes, cellular components, and molecular functions using the Gene Ontology (GO) database, resulting in 33 functional entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis revealed that the transcriptome of P. vannamei had 240 differentially expressed genes annotated across six categories: metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, and human disease. The main metabolic pathways included carbohydrate metabolism, lipid metabolism, protein translation, signal transduction, cell growth and apoptosis, transport and catabolism, immune system, and endocrine system. GO functional enrichment analysis revealed that the functions of significantly differentially expressed genes were primarily enriched in catalytic activity, heterocyclic compound binding, carbohydrate derivatives, small molecule binding, protein folding, and RNA metabolism. Among the top 20 enriched KEGG pathways, the ribosome biosynthesis pathway was significantly enriched. Additionally, pathways related to lipids, atherosclerosis, endoplasmic reticulum protein processing, and purine metabolism were also enriched. The number of differentially expressed genes was more than those of the untreated control. These pathways may be involved in metabolism, environmental information processing, and cellular processes of P. vannamei under microcystin stress. Consequently, the top ten genes with the most significant differential expression were further screened. Notably, zinc finger protein 761-like expression was down-regulated. Differential gene expression statistical analysis indicated that several genes belonging to the zinc finger protein family were significantly down-regulated. This suggests their potential involvement in the microcystin response of P. vannamei. In conclusion, this study provides a methodological reference for shrimp exposure experiments and the molecular regulation mechanism of P. vannamei in response to microcystins. However, the reverse-gavage method requires improvement, and a methodological comparison of different approaches is also necessary to understand the advantages and disadvantages of reverse-gavage methods. Simultaneously, further verification of the differentially expressed genes is required to determine their close relationship with the response of P. vannamei to microcystin stress. These findings will enable in-depth exploration and improvement of the toxicological mechanism in P. vannamei.

Abstract:Boveria disease affects the aquaculture efficiency of sea cucumbers. Boveria labialis causes Boveria disease in Apostichopus japonicus. Owing to the lack of rapid and accurate detection methods, analyzing the transmission route of the pathogen remains complicated. Based on the partial mitochondrial genome sequence of B. labialis obtained using sequencing, primers were designed to amplify nad10 and a SYBR GreenⅠreal-time fluorescent quantitative PCR detection method was established. The concentration of B. labialis in different culture areas and different culture modes of A. japonicus was detected and analyzed. The standard curve established by the designed primers had a good linear relationship in the range of the plasmid standard of 4.05×101–4.05×109 copies/µL, with a reliability (R2) of 0.997. The melting curve showed a single peak, without primer dimers or nonspecific amplification. The minimum detection limit of sensitivity test was 40.5 copies/µL. The designed primers showed specific amplification for B. labialis only and exhibited no cross reaction to Uronema sp., chaenea sp., Colpoda sp. and Paramecium sp. In the repeatability test, the homogeneity of Ct values in the intra-assay and inter-assay of each concentration was high, the CV values of intra- and inter-assay tests were 0.32%–0.82% and 0.40%–0.88%, respectively, indicating good stability. This method was used to detect environmental samples and feeds in different culture areas of four kinds of A. japonicus culture modes. Combined with the comparative analysis of microscopic examination results, there was a moderate positive correlation (R=0.563) between the DNA load of B. labialis in sea water and the infection degree of B. labialis in A. japonicus. A high positive correlation (R=0.931) was found between the DNA load of B. labialis in sediment and attachment samples and the infection degree of B. labialis in A. japonicus. Fresh sea mud was an important carrier for B. labialis transmission. The relevant research results provide reference for the rapid detection, transmission route analysis, and B. labialis prevention and control.

Abstract:Rainbow trout is an important cold-water fish farmed worldwide, and it is cultured on a large scale in Gansu, Qinghai, and Xinjiang Province in China. With the development of rainbow trout farming and increase in production recently, diseases have gradually become an important limiting factor for rainbow trout development, causing huge economic losses, threatening the health of rainbow trout, and sustainable aquaculture industry development in China. The major rainbow trout pathogens are infectious hematopoietic necrosis virus (IHNV) and Aeromonas salmonicida. IHNV is the causative agent of IHN and causes necrosis of the kidneys, spleens, and hematopoietic tissues of fish, with a mortality greater than 90%, causing great economic losses to the rainbow trout farming industry worldwide. The IHNV genome encodes five structural and one non-structural proteins. Among them, the glycoprotein, also known as the G protein, is the only surface protein and main antigen of the virus. G protein stimulates neutralizing antibodies in the host, inducing cellular immunity, and playing an important role in viral pathogenicity and immune responses. Currently, most studies have focused on DNA vaccines targeting the IHNV G protein. A. salmonicida causes furunculosis and ulcers in various fish species, including rainbow trout. Currently, internationally commercialized vaccines against A. salmonicida are widely used, however China still mainly depends on antibiotics for disease control. Therefore, in China, developing a vaccine against A. salmonicida is necessary. Herein, the IHNV-G protein gene was amplified by PCR and ligated into the pGEX-4T-1 plasmid to obtain the G protein expression vector, pGEX-4T-1-G. The recombinant plasmid pGEX-4T-1-G was transformed into A. salmonicida subsp. salmonicida SC18032201 by electronic transformation, to obtain the A. salmonicida vaccine carrier SC18032201-G, which expressed the G protein of IHNV as a polyvalent vaccine. The SC18032201 with pGEX-4T-1 plasmid (SC18032201-pGEX) and wild-type strain SC18032201 were used as negative controls. Isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce G protein in SC18032201-G cells. The G protein expressed by A. salmonicida SC18032201-G was detected by western blotting using mouse anti-His protein serum as the primary antibody and goat anti-mouse serum with HRP as the secondary antibody. The results demonstrated that after IPTG induction, specific reaction bands were detected by the recombinant vaccine carrier SC18032201-G carrying the pGEX-4T-1-G plasmid, but not by SC18032201-pGEX and wild-type A. salmonicida SC18032201, which indicated that the G protein was expressed in A. salmonicida SC18032201-G. The expression of G protein was optimized by adjusting the induction time, IPTG concentration, and culture temperature. Western blotting showed that the best induction condition for recombinant G protein expression by SC18032201-G was 0.2 mmol/L IPTG at 28 ℃ for 8 h. The optimized conditions were used for the incubation and induction of SC18032201-G, and the bacteria were inactivated with formaldehyde. Then the inactivated bacteria were emulsified with Montanide™ ISA 763A VG as adjuvant to prepare an oil-based vaccine. A PBS control was prepared using the same method. Rainbow trout were immunized by intraperitoneal injection with a vaccine or phosphate buffered saline (PBS) control. Forty-five days post-vaccination, 10 rainbow trout from each group were randomly selected for blood sampling from the caudal vertebrae. Blood was stored at 4 °C overnight and centrifuged to obtain the serum for antibody detection. The serum was diluted two-fold (1:2−1:256) and incubated at a 1:1 ratio with IHNV viral culture medium [100× TCID50(50% tissue culture infective dose)]. The mixture was added to a monolayer of carp epithelial tumor cells (EPC) with eight replicates per gradient. The cells were incubated at 15 ℃ and observed for 7 d. The cytopathic effect (CPE) of the culture was recorded, and the highest serum dilution that inhibited 50% of the CPE was recorded as the neutralizing antibody level. The results showed that the neutralizing antibody titer was 54.95±6.76 in the vaccinated group, and no neutralizing antibody potency was detected in the control group. The difference between the neutralizing antibody titers of the immunized and control groups was highly significant (P<0.01). Forty-five days after immunization, rainbow trout were infected with 1×106 CFU/mL A. salmonicida SC18032201 by immersion and observed continuously for 30 d. The mortality of rainbow trout was recorded for 30 d, and the relative survival percentage was calculated. The results showed that the relative survival percentage of rainbow trout vaccinated against A. salmonicida was 100% after 45 d of immunization, which was significantly different from that of the control group. In conclusion, we constructed an A. salmonicida vaccine carrier that expresses the G protein of IHNV, which provides effective protection against A. salmonicida and induces the specific neutralizing antibody of IHNV in rainbow trout. The vaccine carrier can be used as a polyvalent vaccine for major rainbow trout pathogens and as an effective route for disease control in rainbow trout farming in the future.

Abstract:In this study, three efficient probiotic strains, Bacillus subtilis, Enterococcus faecium, and Saccharomyces cerevisiae, isolated from the intestine of Penaeus vannamei, were selected as fermentation strains. This study focused on optimizing the process conditions of compound probiotic fermented feed by orthogonal experiments and investigating the effects on growth performance, digestion, and immunity function of P. vannamei fed with fermented feeds, with the aim of improving the nutritional performance of the compound feed and establishing a fermentation technology for shrimp feed. The results showed that with an inoculation ratio of the three strains of 1%:1%:1%, the process conditions of three efficient fermented feeds (the lowest pH group, the highest acid-soluble protein group, and the highest acid polysaccharide group) were obtained by optimizing the fermentation temperature, water ratio, and fermentation time through orthogonal experiments. During the 28-day feeding experiment, shrimp fed the three fermented feed groups exhibited an increase in survival rate, weight gain rate, and specific growth rate. The lowest pH and highest acid-soluble protein groups had significantly lower feed conversion rates (P<0.05). The hepatopancreatic index of shrimp in the lowest pH group and the highest acid-soluble protein group increased significantly, whereas there was no significant difference in the crude lipid content among the three fermented feed groups. In addition, the protease and amylase activities of the hepatopancreas and intestine in the highest acidic polysaccharide group increased significantly (P<0.05). Compared with the control group, the total hemocyte count and phagocytosis rate were the highest in the highest acidic polysaccharide group, followed by the lowest pH and highest acid-soluble protein groups. The gene expression of Pen3 and Crustin in hemocytes increased significantly, whereas the expression of Pen3, Crustin, and ALF increased significantly in the intestine (P<0.05). However, the expression levels of inflammatory factors did not differ significantly among the three fermented feed groups. In conclusion, the three fermented feed groups, obtained by optimizing the fermentation process of the three probiotic strains, significantly improved the growth performance and immune defense level of P. vannamei. After comprehensive evaluation, the acidic polysaccharide group was found to be the most efficient fermentation feed for P. vannamei.

Abstract:Vibrio alginolyticus is a primary etiological agent for aquatic animal death. V. alginolyticus can infect fish, causing bleeding, ulcer, and blood poisoning; shrimps, causing shrimp post-larvae bacterial vitrified syndrome, acute hepatopancreatic necrosis disease, rotted gill disease, and white fecal syndrome; crabs, causing milk and toothpaste diseases; sea cucumbers, causing skin ulcer syndrome; and shellfish, thereby leading to substantial economic loss in the marine aquaculture industry. Antibiotics, disinfectants, and microecologics are usually used in the aquaculture to prevent or cure such infectious diseases however, their outcomes remain unsatisfactory. In addition, irrational drug use increases risks, such as environmental pollution, bacterial drug resistance, and drug residue. Compared with antibiotics, traditional Chinese medicines (TCMs) present certain advantages, such as antibacterial properties, immunoregulation, slight toxic and side effect, and low drug resistance or residue. Therefore, TCMs have garnered increasing attention in recent years. Using V. alginolyticus and V. parahemolyticus isolated from penaeid shrimp larvae with bacterial vitrified syndrome as research objects, we screened the bacteriostatic activity of 50 TCMs. Four kinds of TCM, namely Terminalia chebula, Galla chinensis, pomegranate peel, and Sanguisorba officinalis, demonstrated good bacteriostatic effects. Gallic acid (GA) is a main active component of these four TCM compounds. GA has good antibacterial, antiviral, anti-inflammatory, and antioxidative properties and can protect the liver and improve the immunological function of the body, thus it may be used to treat and prevent multiple animal diseases. Although GA has antibacterial effects on various bacteria, the bacteriostatic activity and possible mechanism against V. alginolyticus remains unelucidated. By measuring the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and growth curve of GA against V. alginolyticus, we evaluated the bacteriostatic activity of GA against V. alginolyticus. Moreover, the changes in the AKP activity and electrical conductivity of the supernatant fluid of bacteria solution and biofilms, moveability, and aggregation capacity of V. alginolyticus were determined before and after GA treatment to investigate the bacteriostatic mechanism of GA against V. alginolyticus. The MIC and MBC of GA against V. alginolyticus were 4 mg/mL and 8 mg/mL, respectively. These two GA concentrations could completely inhibit V. alginolyticus growth, while 2 mg/mL GA significantly suppressed V. alginolyticus growth, suggesting that the inhibitory effect of GA on V. alginolyticus was dose-dependent. Thus, GA concentrations greater than 4 mg/mL should be selected and used to achieve an effective bacteriostatic effect on V. alginolyticus. The bacterial cell wall is an important structure that maintains cell morphology and facilitates cell protection. Biofilms comprise various extracellular materials, such as proteins, exopolysaccharides, lipids, and extracellular DNA. Biofilms not only enhance the resistance of bacteria to adverse external environment, but also increase their resistance to antibacterial agents. However, TCM can restrain bacterial biofilm formation and development, destroy their cell wall and membrane, affect protein and nucleic acid synthesis, promote oxidative stress, and inhibit virulent factor expression to ultimately suppress or kill bacteria. The changes in AKP and electrical conductivity of bacterial culture serve as an index to verify whether the bacterial cell wall was destroyed or whether its permeability increased. GA (1, 2, 4, and 8 mg/mL) destroyed the cell wall within 2 h and caused AKP leakage. Furthermore, the degree of V. alginolyticus cell wall destruction was positively correlated with GA concentrations; 2, 4, and 8 mg/mL GA could remarkably increase electrical conductivity of the supernatant fluid of V. alginolyticus; compared with the positive control group, 4 and 8 mg/mL GA had a significant inhibitory effect on the formation of the biofilms of V. alginolyticus, with an inhibition ratio of 83.26% (P<0.05) and 77.80% (P<0.05), respectively. Meanwhile, 4 and 8 mg/mL GA notably eliminated mature biofilms, with an elimination ratio of 68.01% (P<0.05) and 67.54% (P<0.05), respectively; 4 and 8 mg/mL GA completely suppressed V. alginolyticus growth on LB swimming motility agar plates; and 1, 2, 4, and 8 mg/mL GA significantly inhibited V. alginolyticus aggregation capacity, with aggregation rates reduced to 18.68% (P<0.05), 19.19% (P<0.05), 25.70% (P<0.05), and 37.41% (P<0.05), respectively. In conclusion, GA has a strong inhibitory effect on V. alginolyticus by growth restraint, cell wall destruction, cell membrane permeability increase, biofilm formation suppression, mature biofilm elimination, and moveability and aggregation inhibition. This study presents a foundation for exploring the action mechanism of GA in suppressing V. alginolyticus growth and provides a theoretical basis for GA in preventing and curing infectious diseases caused by V. alginolyticus in aquatic animals.

Abstract:Ammonia nitrogen and nitrite nitrogen accumulation often occur in industrial whiteleg shrimp (Penaeus vannamei) farming and has a negative impact. To explore the role of microorganisms in ammonia nitrogen and nitrite nitrogen accumulation, water samples from industrial farming systems of P. vannamei were randomly collected and divided into four groups. The four groups were as follows: DG group, ammonia nitrogen<1.33 mg/L and nitrite nitrogen<0.77 mg/L; DB group, ammonia nitrogen>2.53 mg/L and nitrite nitrogen<0.77 mg/L; DY group, ammonia nitrogen<1.33 mg/L and nitrite nitrogen>2.55 mg/L; DR group, ammonia nitrogen>2.53 mg/L and nitrite nitrogen>2.55 mg/L. 16S rRNA sequencing technology was used to analyze the microbial structure of each group, real-time quantitative PCR was used to determine the absolute abundance of nitrogen cycling genes, and Person correlation analysis was conducted for microbial abundance, environmental factors, and nitrogen cycling gene abundance. The relative abundances of Rhodobacteraceae in the DY and DR groups were higher than those in the DG and DB groups, while that of Stappiaceae was lower than that in the DG and DB groups. The relative abundance of norank_o__PeM15 was significantly higher in DG group than that in the other three groups (P<0.05). Among bacteriaceae, Cyanobiaceae, Saprospiraceae, and Cryomorphaceae were significantly positively correlated with ammonia nitrogen, whereas Microbacteriaceae was significantly positively correlated with nitrite nitrogen (P<0.05). The absolute abundances of nitrogen cycling functional genes in DR Group were the highest, and the absolute abundances of narG, nirS, nirK, amoA, and ureC were significantly different from those in other groups (P<0.05). Among functional genes, the abundance of amoA was positively correlated with ammonia nitrogen and nitrite nitrogen, whereas nirS was positively correlated with nitrite nitrogen (P<0.05). These results suggest that the variation of the abundances of Saprospiraceae, Cryomorphaceae, and Microbacteriaceae may affect ammonia nitrogen and nitrite nitrogen concentrations in water. The absolute abundance of nitrogen cycling genes in water with high ammonia nitrogen and nitrite nitrogen concentrations was high. Our study reveals the relationship between water microorganisms, nitrogen cycling genes, and nitrogen-containing compounds, and provides theoretical support for addressing harmful nitrogen accumulation in shrimp industrial farming via microorganism regulation.