Abstract:

Abstract:

Abstract:The Anqing section of the Yangtze (Changjiang) River in Anhui Province, China, is one of the most important migratory routes and distribution of the highly valued diadromous estuarine tapertail anchovy Coilia nasus. In the past, this section has suffered a severe loss of C. nasus resources caused by human activities, especially overfishing and water pollution. Since 2019, a fishing ban policy for the species in the Yangtze River has been introduced in China for resource restoration. Otoliths are calcium carbonate structures, and otolith microchemistry can provide detailed life history information that can be used to compare habitat use between different saline water bodies. To study the habitat history of C. nasus in the Anqing section of the Yangtze River after the implementation of the fishing ban, we examined the microchemical characteristics (Sr and Ca) of otoliths from the long (previously named C. ectenes) and short maxillary (previously named C. brachygnathus) types of anchovy using electron probe X-ray microanalysis (EPMA). Line-transect analysis of Sr/Ca ratios suggested that the short maxillary type of C. nasus could be divided into two groups. The otolith Sr/Ca ratios (calculated and expressed as Sr/Ca ×1,000) of one group were always < 3.0, indicating their freshwater residency. Those of the other group had phases of low (< 3.0, believed to be indicative of a freshwater habitat) and high values (> 3.0, believed to be indicative of a brackish or seawater habitat), indicating that anchovies not only have a freshwater habitat history with low salinity but also a brackish habitat history with high salinity. The latter group of anchovies were typically anadromous C. nasus, as the Sr/Ca ratio of the otoliths of C. nasus fluctuated significantly and coincided with variations between freshwater and estuarine brackish or seawater (i.e., anchovies experienced not only freshwater habitats but also brackish water habitats at different stages of their life history). The Sr content mapping of EPMA with different color patterns for freshwater (blue), brackish water (green-yellow) and seawater (red) habitats also confirmed the results obtained from the line-transect analysis. Our results demonstrated that the population composition of C. nasus in the Anqing section of the Yangtze river became complex, and there was coexistence of freshwater resident and anadromous C. nasus in the Anqing section of the Yangtze River in Anhui Province, China (i.e., freshwater resident and anadromous short maxillary and anadromous long maxillary type C. nasus). This phenomenon may suggest a restoration of C. nasus resource diversity after the implementation of the fishing ban in the Anqing section of the Yangtze River.

Abstract:The leopard coral grouper (Plectropomus leopardus) belongs to the family Epinephelinae, and genus Plectropomus. Vibrio harveyi is the main pathogen that causes "rot disease" in leopard coral grouper, which is a major threat to the sustainable development of its aquaculture industry. The disease is highly prevalent from June to August and severely affects aquaculture. Therefore, developing disease-resistant strains is a necessity. However, currently, artificial breeding techniques for leopard coral groupers cannot establish a family lineage through one-on-one artificial insemination, making traditional breeding methods that depend on a clear pedigree difficult. Considering the successful breeding of disease-resistant fish species with or without a pedigree, genome selection breeding technology are vital for cultivating disease-resistant leopard coral groupers. In genetic selection, the genetic parameters of target traits are important reference factors for specifying breeding programs. To evaluate the genetic parameters of leopard coral grouper resistance to V. harveyi, we constructed a genome-relatedness matrix based on high-density single-nucleotide polymorphisms using four models (binary linear model [BLM], binary threshold model [BTM], longitudinal linear model [LLM], and linear threshold model [LTM]) to fit two disease-resistant phenotypes (test-day trait, TDS; Bivariate survival trait, TS), and used restricted maximum likelihood [REML] to estimate variance components. Our findings illustrated that the genetic heritability of leopard coral grouper resistance to V. harveyi ranged from 0.182 to 0.486, which belongs to the medium-to-high genetic heritability range. The additive genetic variance ranged from 0.071 to 0.262. The genetic heritability estimated by the linear model was 0.382 and 0.476, whereas that estimated by the threshold model was 0.182 and 0.207, respectively. These results suggest that leopard coral groupers resistance to V. harveyi can be improved through genetic breeding. Herein, the linear models (BLM and LLM) obtained higher genetic heritability estimates and more accurate genomic estimated breeding value (GEBV) predictions than the threshold models (BTM and LTM). However, despite the model used, the correlation coefficient between the GEBV rankings under the same phenotype definition >0.9, indicating that their impact on the GEBV ranking was not significant. Compared to the cross-sectional models (BLM and BTM), numerous leopard coral grouper GEBVs were rearranged in the LLM results. There was a strong correlation between the LTM and phenotype (TS), indicating that LLM has an excellent prediction effect. Therefore, when breeding leopard coral groupers for V. harveyi-resistant traits, a LLM should be considered. The study observed that using longitudinal models (LLM and LTM) to estimate genetic heritability produced higher results than the cross-sectional models (BLM and BTM), which may be due to the death time explaining different components of fish disease resistance. In longitudinal models, the genetic component influenced by the time of death is effectively harnessed. However, in cross-sectional models, this effect is inadvertently subsumed within the residuals. Consistent with the genetic heritability findings, the longitudinal models produced more precise GEBVs compared to cross-sectional models. Our results suggest that TDS might offer a more accurate measure for assessing the resistance of leopard coral groupers to V. harveyi than the TS. Compared with the threshold models, linear models performed better in GEBV prediction, and higher genetic heritability estimates were obtained. Although, most previous studies on disease resistance traits have reported inconsistent genetic heritability estimates between threshold and linear models, some studies support these conclusions. This result may be due to differences in information processing between the different models, which leads to different results. In this study, the additive genetic variance obtained using threshold models (BTM and LTM) was 0.222–0.262, and additive genetic variance obtained using linear models (LLM and BLM) was 0.071–0.086. It is expected that the additive genetic variance obtained using threshold models was higher than that obtained using linear models. Furthermore, the residual variance resulting from fitting linear models was notably low. We posit that when threshold traits are erroneously treated as normally distributed data and linear models are employed for analysis, the residual variance may be underestimated. This underestimation is likely due to the model's underfitting, which consequently leads to an inflated heritability estimate for the linear model. This study aimed to estimate the genetic parameters of leopard coral grouper resistance to V. harveyi using infection test data of leopard coral groupers injected with V. harveyi and to construct an individual genotype relationship matrix based on single-nucleotide polymorphisms. The genetic heritability of leopard coral grouper resistance to V. harveyi was estimated to be between 0.182 and 0.486 by comparing different models and phenotype definitions. The linear (0.382 and 0.476) and threshold models (0.182 and 0.207) were used to estimate genetic heritability. The estimated genetic heritability was within the medium genetic heritability range. Our findings were used to improve the target traits of leopard coral groupers, specifically their resistance to V. harveyi. This study supplements the genetic parameter estimation of leopard coral grouper resistance to V. harveyi and provides a reference for selecting V. harveyi-resistant leopard coral groupers for breeding.

Abstract:The Chinese tongue sole (Cynoglossus semilaevis) belonging to Pleuronectiformes, Cynoglossidae, Cynoglossus, is distributed in the sea areas of Korea, Japan and China. C. semilaevis has limited natural resources and no long-distance migration, which is suitable for the development of aquaculture in coastal areas. After more than ten years of artificial domestication, C. semilaevis has become one of the main mariculture species. Water temperature is an important environmental factor affecting the growth and development of fish. High temperature in summer can cause stress and even death of C. semilaevis, which is one of the important factors affecting factory farming and the promotion in the southern coast. However, the physiological and molecular changes of C. semilaevis in response to acute high temperature stress was still unclear to date. When fish are stimulated by high temperature, it can cause oxidative stress, resulting in a variety of toxic effects. It has been found that there is a significant correlation between high temperature and the activity of antioxidant enzymes in many aquatic animals. Therefore, changes in the activities of antioxidant enzymes can be used as an important indicator when fish are in a state of oxidative stress under high temperature stress. As a tissue related to detoxification metabolism and immunity of fish, liver plays an important role in response to high temperature stress, thus the tissue structure and apoptosis are also important indicators of liver health status after high temperature stress. Heat shock proteins (HSPs) are a class of common biological stress proteins, which have biological functions such as anti-stress, anti-oxidation, and regulation of apoptosis. HSPs are also involved in the resistance of fish to heat stress. Recently, it has been found that zebrafish (Danio rerio) is extremely sensitive to temperature changes after knockout of dual-specificity phosphatase 1 (dusp1) gene, and dusp1 has the possibility of maintaining redox homeostasis, so it was speculated that dusp1 gene may plays an important role in fish resistance to heat stress. In this study, heat shock protein family A member 1A (hspa1a) in the heat shock protein HSP70 family, heat shock protein 90 beta family member 1 (hsp90b1) in the HSP90 family, and dusp1 gene were selected to study the temporal expression characteristics under high temperature stress. In order to explore the effects of high temperature stress on physiological and molecular changes in the liver of C. semilaevis, a full-sib family of C. semilaevis was selected as the experimental object to detect the oxidative damage and heat stress-related gene expressions. After continuous heating to high temperature stress conditions (35 ℃), liver tissues were collected at 0 h, 3 h, 6 h, 12 h and 24 h, respectively. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to observe cell damage, antioxidant enzyme activity and malondialdehyde (MDA) content were measured, and the expression changes of stress-related genes hspa1a, hsp90b1 and dusp1 were detected. The results showed that acute high temperature stress could cause obvious pathological changes and apoptosis in the liver tissue of C. semilaevis. The activity of antioxidant enzyme superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and the content of MDA in the high temperature stress group was significantly higher than that of the control group at 6 h, 12 h, 0 h and 24 h, respectively (P<0.05). The expression of heat shock protein genes hspa1a and hsp90b1 were significantly up-regulated at 0 h and 3 h after high temperature stress, respectively. Heat stress-related gene dusp1 was significantly up-regulated at 3 h after high temperature stress. In conclusion, oxidative stress occurs in the liver of C. semilaevis under acute high temperature stress, in the short term, the body can mobilize the antioxidant system to accelerate the removal of reactive oxygen species and activate the expression of heat stress-related genes. This study provides a reference for analyzing the response mechanism of C. semilaevis to high temperature stress, preventing the occurrence of massive death in summer, and carrying out the breeding of high temperature resistant varieties.

Abstract:Chinese tongue sole (Cynoglossus semilaevis) is a traditional, nutrient rich and economically valuable fish that is important for mariculture in China. It has small visceral masses, few spines, with tender and tasty flesh. Studies on the sexing mechanism of fish were carried out since the revelation of sexual reversal and hermaphroditism in the eel. Sex is the most common biological phenomenon and there are significant differences in morphology, reproductive strategies, and behavior between females and males of many organisms. Sole is a typical representative of the heterogeneous growth of males and females: adult females are approximately 2–4 times larger than males. This has constrained the sustainable development of the sole culture industry. Why do females grow faster than males? Do sex differentiation-related genes play a role in sex determination and size heteromorphism in sole? Answering these questions has important implications and applications in the genetic improvement and development of sole farming. Rimoc1 encodes a protein in the inner mitochondrial membrane that is involved in regulating the activity of complex Ⅰ of the mitochondrial respiratory chain that affects mitochondrial respiratory function. Rimoc1 interacts with the NDUFAF1 subunit of complex Ⅰ in the inner mitochondrial membrane and enhances its affinity for NADH. This increases the catalytic activity of complex Ⅰ and increases the rate of electron transfer in the mitochondrial respiratory chain. In addition, RIMOC1 interacts with proteins in other mitochondrial respiratory chain complexes (such as complex Ⅲ and complex Ⅳ) and may play a role in regulating the overall stability and function of the mitochondrial respiratory chain. This study aimed to investigate the expression differences of the rimoc1 gene in male and female Chinese tongue sole and determine its potential relationship with sex and growth. Rimoc1 expression was highest in the ovaries and muscles of females; it was not expressed in male tissues. Interestingly, its expression was relatively stable in the ovaries, but increased at 1.5- and 3-years post-hatching. Knocking out rimoc1 through siRNA interference in cell lines significantly reduced the expression levels of growth-related gene igf1, and sex differentiation-related genes sox9b and foxl2, while sox9a expression increased. Promoter activity analysis further demonstrated that the rimoc1 promoter strongly responded to transcription factors C/EBPdelta, Sox2, and c-Jun. This suggested their potential role in regulating rimoc1. Sex determination in semislipped sole occurs at approximately 50 days, while gonadal differentiation begins at 60 days. However, differentiation at the cellular level is relatively delayed, with ovarian cell differentiation usually occurring at 90–120 days with the emergence of an ovarian cavity followed by continued oocyte differentiation and sexual maturity at approximately 2 years of age. Thus, high rimoc1 expression at 90 days is consistent with the differentiation of naturally growing ovarian cells in semi-smooth tongue sole. Analysis of promoter activity showed that rimoc1 was transcriptionally active at 981 bp upstream and had strong initiation activity. The activity was enhanced following cotransfection with three transcription factors (C/EBPalpha, Sox2, and C-Jun), although only the C/EBPalpha group was significantly different. This suggested that these three transcription factors exert an enhancing effect on rimoc1 transcription. It is known that C/EBPalpha plays a major role in keratinocyte and adipocyte differentiation. However, its regulation of rimoc1 in hemipterous sole requires further investigation. There are few reports on the function of rimoc1 in sexual differentiation. Therefore, we performed the first functional investigation of rimoc1 by siRNA-mediated RNA interference in an ovarian cell line. Quantitative polymerase chain reaction showed that the expression levels of igf1, sox9a, sox9b, and foxl2 changed at different degrees after rimoc1 gene interference compared to the control group; sox9a expression was up-regulated, while the expression of igf1, sox9b, and foxl2 was down-regulated. Fish igf1 is closely related to reproductive function and gonadal development. Tilapia igf1 expression was detected from the early stages of oogenesis till oocyte maturation. At the same time, Igf1 is involved in regulating growth hormones and growth. Sexual growth dimorphism begins to appear in one-year old half-sauropod sole. The quantification of different developmental stages shows that rimoc1 expression starts to increase at 180 days until its peak expression at 1.5 years of age. It is worthwhile investigating whether rimoc1 has a potential regulatory function in growth. Sox9 is a key transcription factor that plays an important role in regulating the proliferation and differentiation of various cell types. It has a critical role in embryonic development, with expression occurring throughout this period. Its level is maintained in adult tissues and plays an important role in the repair of postnatal damage in endodermal and ectodermal organs. It has a role in sex differentiation, with sox9a and cyp19a together forming a regulatory pathway for sexual differentiation. We hypothesize that rimoc1 is involved in ovarian development and oogenesis by regulating the steroid hormone pathway. Sox9b is highly expressed in the gonads of early semi-smooth sole, and rimoc1 knockdown results in its reduced expression. This suggests that Rimoc1 and Sox9b act together to regulate Foxl2 and play a role in sex determination and differentiation, ovarian development and maintenance, embryonic development, and immune regulation in the animal. The foxl2 gene positively regulates ovarian differentiation in mice, and its sustained expression inhibits abnormal differentiation of ovarian cells to testicular cells during growth and development, thereby maintaining the female sex. Meanwhile foxl2 deletion can lead to poor ovarian development, and even female sterility. Rimoc1 knockdown results in its reduced expression, suggesting that rimoc1 may be closely associated with ovarian development in hemipelagic sole. Therefore, a deeper understanding of the function and regulatory mechanisms of rimoc1 may be the key to resolving sexual growth dimorphism. Our results suggest that rimoc1 plays a crucial role in sex differentiation and growth of Chinese tongue sole. These findings provide a foundation for further investigations into the sexual growth dimorphism of this species.

Abstract:Rainbow trout (Oncorhynchus mykiss) is one of the world's most widely farmed cold-water economic fish. It is also the primary cold-water fish species in China. Since the 1960s, the rainbow trout germplasm has been introduced from North Korea, the United States, Denmark, and other countries, and a systematic study has been carried out on germplasm preservation, identification, breeding, and variety breeding. Compared with rainbow trout diploid, the triploid has the advantages of a high feed conversion rate, fast growth rate, and good meat quality. Moreover, the triploid gonadal hypoplasia can mitigate risks associated with high mortality and meat quality decline during spawning and avert the ecological risk of invasive alien species caused by breeding escape. Therefore, breeding triploid rainbow trout has more significant market and ecological benefits. As diploid and triploid rainbow trout are very similar in terms of morphology, it is difficult to distinguish the ploidy of rainbow trout by morphology alone. Currently, DNA content analysis by flow cytometry is the main method used to identify the ploidy of rainbow trout, but this method necessitates the collection of fish blood or tissue samples, and the body length of the fish to collect the blood must be at least 5 cm. Additionally, these sampling operations may harm the fish. This method also involves a delicate operation, complicated technical process, and expensive consumables, thus, the popularization and application of triploid identification of rainbow trout are greatly restricted. Considering the limitations of the technical test for the ploidy of rainbow trout, a minimally invasive and economical method with only a small amount of sampling is needed to identify and analyze the ploidy of rainbow trout in batches. Microsatellite markers (SSR markers) are simple repeated sequences widely distributed in eukaryotic genomes and have been widely used in ploidy and pedigree analyses of fish. The advantages of microsatellite (SSR) analysis include high reproducibility, low sample requirements, and rapid population genetic analysis. In order to achieve the use of a minimally invasive method to collect samples for trout ploidy identification, this study analyzed 153 SSR markers using PCR amplification and electrophoresis separation technology. Among them, 139 SSR markers could be successfully amplified in diploid and triploid, and 132 SSR markers were screened for polymorphism. Finally, seven markers showed high variability in triploid. By verifying 52 reference samples with a known ploidy level and 48 samples with an unknown ploidy level, three SSR markers (SSR1054, SSR1056, and SSR1468) were further screened to distinguish the ploidy level. Moreover, three pairs of primer sequences with good stability were redesigned according to the three marker sequences for ploidy analysis application. When SSR markers were selected for ploidy identification, amplification sites with high variability were preferred because they greatly improve the efficiency of SSR-assisted ploidy identification. Differentiation of the variability of amplification sites mainly considers the number of alleles expressed and the relative frequency of these alleles. Furthermore, having clear map bands that are easy to observe and analyze is an important criterion for screening high-quality specific SSR markers. Statistical clear map bands can correctly judge the allelic configuration at each locus, thus, determining the ploidy level of rainbow trout. However, when the number of microsatellite DNA alleles shown in the electrophoretic map was less than the maximum number of alleles that may occur at the ploidy level of the polyploid species, it was difficult to accurately judge the ploidy by direct observation of the number of alleles. In diploid individuals, highly heterozygous SSR amplifies at most two alleles at a given site. Similarly, three and four alleles can be observed in triploid and tetraploid individuals, respectively. However, in some rare cases, the actual number of alleles cannot be correctly determined by looking directly at the number of alleles. For example, individuals with unknown ploidy show multiple possible genotypes for two alleles, which can occur with both diploids (AB) and triploids (AAB or ABB). Therefore, this study combined the analysis results of multiple specific SSR markers to improve the accuracy of ploidy detection. In addition, this study found three specific labeling bands that can assist in the differentiation of diploid and triploid rainbow trout, namely, SSR1054-305bp, SSR1056-463bp, and SSR1468-363bp. These three specific markers will greatly improve the accuracy of using SSR markers to identify triploid rainbow trout. Microsatellite null alleles are another potential limitation in SSR ploidy identification, which can be eliminated or avoided by changing the binding site of primers and flanking sequences due to the failure of normal gene amplification due to mutations in SSR lateral sequences. Although the results of this study cannot eliminate the presence of null alleles in future samples, the present experimental data suggest that the frequency of null alleles is so low that their impact on polyploid identification is minimal. SSR markers screened in this study solve the existing problems of a complex technical process and expensive consumables in the identification of rainbow trout ploidy, providing a minimally invasive, economical, reliable, and batch-operable molecular method and contributing to the study of the genetic diversity of rainbow trout with different ploidy.

Abstract:Thamnaconus septentrionali has excellent breeding characteristics, is omnivorous, easy to domesticate, is especially suitable for netting, cleans the netting, and effectively reduces labor-costs. T. septentrionali is available in various sizes and is raised in the market at 100 g. Under water temperature of 18–25 ℃, after 5–6 months of breeding, it reaches commercial size. As the Yellow Sea and Bohai Sea are cold in winter, a suitable breeding cycle for green fins is from May to November annually. Under natural conditions, the spawning period of T. septentrionali in the Yellow Sea and Bohai Sea is from early May to early June, making good use of the suitable breeding cycle of net box culture. Therefore, reproductive regulation is required to advance the reproductive period of T. septentrionali. Previous studies have primarily focused on the expression and functional analysis of individual genes. In contrast, no studies exist on the overall expression analysis of gonad-related genes based on histology. Herein, we conducted the first transcriptome sequencing analysis of the spermatophores and ovaries of T. septentrionali using an Illumina high-throughput sequencing platform. Overall, 165,981,523 raw reads were sequenced from the cDNA, and 161,234,846 clean reads were obtained after quality control. The Q20 of each sample was >98.43%, Q30 was >95.25%, and GC content of the sample bases was greater than 52.31%. Our findings indicate that the sequencing data are accurate, of good quality, and were used for subsequent analyses. The obtained unigenes were annotated in the NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), PFAM (protein family), KOG (euKaryotic Ortholog Groups), SwissProt (a manually annotated and reviewed protein sequence database), GO (Gene Ontology), and KO (KEGG Orthology) databases, and 24,009, 35,057, 18,453, 26,971, 30,294, 11,420, and 21,613 unigenes were annotated, respectively. The KEGG annotation results were divided into five major categories: organic systems, with 2,115 genes in nine pathways; metabolism, with 1,444 genes in 12 pathways; environmental information processing, with 1,200 genes in three pathways; and genetic information processing, with 1,128 genes in four pathways. Environmental information processing (1,200 genes in three pathways), genetic information processing (1,128 genes in four pathways), and cellular processes (1,128 genes in four pathways). The number of genes enriched in cellular process-related pathways was 927 distributed in four related pathways. The signal transduction pathway was the most annotated KEGG pathway with 804 genes. The experiment used 24,546 unigenes annotated in the KOG database, which were then classified into 26 categories based on their function. The largest number of unigenes were annotated in the signal transduction mechanism category (5,411), followed by the general function prediction-only category (4,252 unigenes), and posttranslational modification, protein folding and chaperone category (1,713 unigenes). Unigene posttranslational modification, protein turnover, chaperones; transcription, 1,613; and other related functions. There were 18,954 differentially expressed genes (DEGs) in the spermatophore and ovary transcriptomes of the T. septentrionali, with 11,265 genes up-regulated and 7,689 down-regulated in the ovary relative to the spermatophore. The GO functional enrichment analysis revealed that DEGs were most enriched in the cellular component of biological processes, intracellular component of the cellular component subclass, and nucleic acid binding of the molecular functional subclass. GO analysis of the DEGs in males and females provided a partial list of genes associated with the reproductive process (GO: 0022414), sexual reproduction (GO: 0019953), gamete formation (GO: 0007276,), sex differentiation (GO: 0007548), and gonad development (GO: 00084060). To further characterize the specific functions of the DEGs in the spermatophores and ovaries of T. septentrionali, the enriched signaling pathways were further analyzed using the KEGG database. In total, 154 KEGG pathways were included, of which the top 30 were selected. The functional pathways with the highest number of expressed genes were closely associated with gonadal sex differentiation and development, including the insulin signaling pathway, steroid hormone biosynthesis, FoxO signaling pathway, M-TOR signaling pathway, and progesterone-mediated oocyte maturation. Among these, the FoxO signaling pathway regulatory genes are expressed in multiple processes, such as cell cycle control, apoptosis, and gluconeogenesis. The insulin signaling pathway plays an important role in regulating developmental, metabolic, and lifespan physiological processes, and the insulin signaling pathway is involved in gonadal development and maturation. Nine DEGs, bmp2, sox3, figla, hsd17b1, cyp19a, cyp17, foxl2, star, and amh, were selected for real-time fluorescence quantitative PCR (qRT-PCR) validation. The qPCR results were consistent with those of RNA-seq analysis. GO and KEGG enrichment results were analyzed and revealed that amh, cyp17, and star are imperative in male spermatogenesis of T. septentrionali; bmp2, foxl2, cyp19a, figla, and hsd17b1 are essential in female oogenesis and ovarian steroidogenesis. By comparing the transcriptome expression differences between the spermatophore and ovary of T. septentrionali, we elucidated the gene expression characteristics of the spermatophore and ovary, laying the foundation for future research on the mechanism of reproductive development of T. septentrionali and providing theoretical support for optimizing reproductive regulation techniques.

Abstract:Fish skin is a multipurpose tissue with important functions—protection, perception, and hormone metabolism—in the fish body. As a byproduct of fish processing, it can be used as a material for extracting collagen, making leather, and related pathological research. However, there have been few reports on the tissue structure of fish skin. This study aimed to analyze the structural characteristics of Thamnaconus septentrionalis fish skin in order to provide a reference for the utilization of its resources. Six T. septentrionalis samples were selected and their skins were observed using frozen sectioning, skeletal, hematoxylin & eosin (H&E), Periodic acid-Schiff (PAS), Masson, Van Gieson (VG) staining, and scanning electron microscopy. The images obtained by slice scanning were analyzed using SlideViewer (3DHISTECH), ImageJ, and Photoshop software. The SEM images were analyzed using ImageJ and Photoshop software. SPSS25.0 software was used to analyze the data of epidermal and squamous layers, conical bone bulge, basal layer, and dermis thicknesses. The results showed that the histological structure of the skin comprised four parts: epidermis, scale layer, dermis, and subcutaneous tissue. The thickness of the epidermal layer was (26.81±7.48) μm. This layer was mainly composed of epithelial and basal cells but also contained a large number of mucus cells. The scale layer consisted of the spine of conical bone and a substrate. The thickness of this layer was (22.49±5.19) μm, and there were two to four rows of the spine of conical bone with different diameters, different bending degrees at the top, and a height of (257.13±10.41) μm unevenly distributed on the base plate. The average thickness of the dermis was (176.97±21.11) μm, and the dermis thickness of the head was lower than that of the middle part of the body, but higher than that of the tail, mainly composed of collagen fiber. The subcutaneous tissue layer was mainly comprised of collagen fibers and non-fibrous stroma. In this study, the histological pattern of the skin of T. septentrionalis was mapped according to its characteristics, providing valuable information for fish skin resource usage.

Abstract:Fish are generally classified as herbivorous, carnivorous, omnivorous, or filter-feeders according to feeding habits, including the feeding method and food content. The feeding habits of fish are reflected in their digestive organ, mainly the intestine. The intestine tract is the main site for digestion and nutritional uptake, and it is regarded as a key organ in fish nutrition. Reportedly, fish gut microbiota contributes to digestion and affect gastrointestinal tract development as well as the overall growth of the fish. The disruption of the intestinal microbiota induced by their feeding habit via diet usually affects the digestive functions of the host through disturbance in bacterial digestive enzyme production. Feeding habits determine the feed composition, which shapes the microbial communities in fish. For example, diet has been reported as a dominant cause of variation in the microbiota composition of rainbow trout. In contrast, feeding habit, which is regulated by many factors, including intrinsic and external factors, such as appetite, structure of the digestive tract, and feed palatability, is an important area of research in the intestinal microflora study. The feeding habit of fishes is one of the main factors affecting the differential colonization of fish intestinal flora. For example, the feeding habit increases the colonization of fish intestinal flora in the order omnivorous > herbivorous > carnivorous. It is also known that feeding habits influence intestinal digestion and structure. However, the influence of feeding habit on proximate composition, digestive enzyme secretion, and intestinal histomorphology is currently not well-known in fish. On the contrary, the feeding habit affects the composition and diversity of the intestinal microflora of fish. The composition and diversity of the microbiota in Carassius auratus, Ctenopharyngodon idella, Siniperca chuatsi, and Schizothorax grahami from the same environment have also not previously been studied. The present study aimed to compare and analyze the differences in the composition and diversity of the intestinal microflora of four fish species, investigating the relationship between their feeding habits and intestinal microflora as well. This study collected 12 intestine samples from each of the four fishes with different feeding habits, including omnivorous (C. auratus), herbivorous (C. idella), filter-feeder (S. chuatsi), and carnivorous (S. grahami), from the same pond. The composition and diversity of the microbial communities were determined by using high-throughput sequencing of 16S rRNA. Meanwhile, this study investigated the effects of feeding habits on the composition and diversity of the intestinal microflora of fish and predicted the potential functions of specific microflora with different nutrients. The results showed that the diversity of the intestinal microflora in fish was significantly affected by feeding habit (P<0.05), such that higher levels occurred in the order omnivorous (C. auratus) > herbivorous (C. idella) > filter-feeder (S. grahami) > carnivorous (S. chuatsi). Proteobacteria and Firmicutes were the common dominant flora among the four fishes, although there were differences at the genera and species levels. For example, Acinetobacter and Bacteroides species were the most dominant flora in S. chuatsi and C. idella, respectively. Function prediction showed that the intestinal tract of S. chuatsi was mainly gram-negative. The disease resistance potential of C. idella was slightly higher than that of the other three fish species. Acinetobacter johnsonii, Acinetobacter lwoffii, and Pseudomonas stutzeri might help the host to digest protein, while Bacteroidetes may help the host to digest cellulose. In conclusion, feeding habit is one of the main factors affecting the differential colonization of fish intestinal flora. Analyzing the correlation between feeding habits and dominant intestinal flora as well as exploring the function of specific flora, could lay a theoretical foundation for studying the microbial effect of fish nutrition metabolism.

Abstract:Acoustic fish driving technology, as an auxiliary fish passage measure and a non-physical barrier, is based on the use of sound signals to prevent or regulate fish behavior. The purpose of these techniques is to guide the fish away from dangerous areas, such as the water inlets of hydroelectric power stations, spillways, and ship locks, allowing them to easily locate the entrance to the fishway, which would help improve fish passage efficiency. Studying the negative phonotaxis behavior of fish is vital for establishing non-physical barriers using acoustic characteristics. However, there has been little research on verifying the effectiveness of acoustic fish deterrence technology in field environments. Therefore, this study used alternating sound playback to conduct negative phonotaxis experiments on grass carp (Ctenopharyngodon idellus) juveniles to explore their behavioral responses to different sounds. The experimental tank (3.6 m×1.1 m×1.0 m) was created in the waters of Xialao Creek in Yichang City, Hubei Province, with an average water depth of 0.5 m and an average flow rate of 0.06 m/s. The experiment used one single-frequency sound (1 000 Hz) and five complex sounds (fish swimming, engine, short-nosed crocodile call, pile driving, and yacht sounds), with a sound pressure level of (117.69±2.77) dB re 1 μPa. The effectiveness of acoustic fish-repellent technology has been proven, but there are only a few applications in practical engineering. On the one hand, the theoretical knowledge is not comprehensive, and on the other hand, there is a gap between theoretical research and practical engineering. Moreover, there are differences in proton movement (vibration) modes between indoor and natural environments. Compared with fish in an indoor environment, fish in natural waters tend to receive sound signals by proton movement rather than sound pressure. At the same time, the distribution of the sound field in natural and indoor environments also differs; thus, field experiments are necessary for the advancement of acoustic fish-repellent technology. Globally, studies on the negative phonotaxis of fish have mainly been conducted in vitro. Detailed studies using natural open water conditions are insufficient, and further field verification experiments are needed. Therefore, this study conducted experiments in natural open water, compared the sound field changes in the natural and indoor environments, and studied negative phonotaxis behavior by observing reaction time, initial reaction time, average reaction time, phonotaxis speed, movement time ratio, and other indicators. The results showed that when the complex sounds were played, the reaction times, tone trend speed and movement time ratio of grass carp were significantly higher than that of single tone and control group (P<0.001), and the initial reaction time and average reaction time of grass carp were significantly lower than that of single tone and control group (P<0.001). Among the complex sounds, the grass carp stimulated by the yacht sound had the largest response times and speed, while the grass carp stimulated by the fish swimming sound had the smallest response times and speed. In the complex sound, the first response time of grass carp stimulated by yacht sound was the shortest, which was (23.40±5.13) s. The first response time of grass carp stimulated by engine sound was (146.00±7.82) s, which was significantly lower than that of other complex sounds (P<0.05). The average response time of grass carp stimulated by the sound of yacht and pile was (26.52±3.01) s and (28.76±4.07) s, respectively. The average response time of grass carp stimulated by fish swimming sound was (64.76±17.82) s. In the complex sound, the motion time ratio of grass carp stimulated by fish swimming sound was the highest, which was (98.47±0.48)%. The motion time ratio of grass carp stimulated by engine sound was (94.58±0.54)%. There were no significant differences in reaction times, initial reaction time, average reaction time and exercise time ratio between grass carp and control group when playing single frequency tone (P>0.05). The experimental results indicated that the five complex sounds used in this study (fish swimming, engine, short-nosed crocodile call, pile driving, and yacht sounds) all had a deterrent effect on grass carp juveniles. This study not only enriches current knowledge of the negative phonotaxis behavior of fish but also provides a scientific basis for the design and optimization of sound-based fish deterrent facilities in practical engineering.

Abstract:Sinonovacula constricta is one of the four traditional cultured shellfish in China. The salinity in the aquaculture water body is easily affected by tides, seasonal rainfall, and high temperatures, and often fluctuates to different degrees. This affects the physiological activities of S. constricta and causes a series of changes in the structure of the osmoregulation organs, osmotic pressure, ion transport, and free amino acid (FAA) content in the body to adapt to the changes in environmental salinity. Aquatic animals can regulate cell volume and maintain osmotic pressure balance through FAAs. This mechanism has been proven in aquatic animals, such as Meretrix lusoria, Crassostrea gigas, Haliotis discus hannai, Penaeus vannamei, and Portunus trituberculatus. The common FAAs that regulate osmotic pressure in bivalves mainly include Ala, Gly, Pro, and Tau. Whether FAAs play an osmoregulation role in S. constricta, whether their involvement in osmoregulation is similar to that in other shellfish, and what the metabolic pathway is of main FAAs deserve further study. This study explored the changes of osmotic pressure and FAAs in the gill, foot, and hemolymph of S. constricta after salinity stress and analyzed the sequence characteristics, tissue expression, and mRNA expression characteristics after salinity stress and RNA interference (RNAi) of the Sc-CARNS gene, and the changes of alanine and carnosine contents. The osmotic pressure and FAA contents in the gills, foot, and hemolymph of S. constricta under different salinities (5, 20, and 35) were measured by freezing point osmometer and automatic amino acid analyzer. At the same time, the expression of the Sc-CARNS gene in the foot under different salinities (5, 20, and 35) was analyzed by RT-qPCR and RNAi technology. The content of alanine was determined with the shellfish alanine ELISA kit. The content of carnosine was determined by phthalaldehyde colorimetry. The results showed that the osmotic pressure in the gills, foot, and hemolymph of S. constricta significantly decreased within 1–72 h under low salt stress (P < 0.05), while under high salt stress, the osmotic pressure in the gills, foot, and hemolymph reached a steady state within 24 h, which was consistent with the osmotic pressure of external seawater. Compared with the control group, the osmotic pressure in the gills, foot, and hemolymph decreased by 66.7%, 69.7%, and 71.6%, respectively, when the salinity was low. The wet weight of tissues was then increased by 68.3%, 67.5%, and 70.2%, respectively. At the same stress time, the osmotic pressure of each group of S. constricta was salinity 35 > salinity 20 > salinity 5. Under normal salinity, the FAAs with the highest content in the gills, foot, and hemolymph of S. constricta were Gly, Arg, and Gly, respectively. After salinity stress, the content of total free amino acids in all tissues increased significantly with the increase of salinity. The main FAAs with the largest content change in the gills, foot, and hemolymph, respectively, were Ala, Gly, Glu, and Pro; Ala, Gly, Arg, and Tua; and Ala, Ser, Thr, and Gly. Ala was the most variable FAA in all tissues. According to the transcriptome results of S. constricta after salinity stress, it was speculated that Sc-CARNS is related to osmotic regulation. The expression of Sc-CARNS was the highest in the muscle type tissues of S. constricta, followed by the gills, and was the lowest in the hepatopancreas. After low salt stress, the expression of the Sc-CARNS gene mRNA in S. constricta increased at first and then decreased with the stress time, and was significantly higher than that in the control group after 4 h of stress (P < 0.01), and reached the peak at 24 h. The content of alanine decreased significantly with time, and the content of carnosine increased significantly with time (P < 0.05). After high salt stress, the expression of Sc-CARNS decreased, and there was no significant change compared with the control group. The content of alanine first increased, then decreased, and then increased after 8 h, and was not lower than that of the control group. The content of carnosine showed a decreasing trend after 24 h. After RNAi, the expression level of Sc-CARNS mRNA in the interference group decreased first and then increased with the interference time under normal salinity. At 24 and 48 h, the expression level of Sc-CARNS mRNA was significantly lower than that of negative control (P < 0.05), and the interference efficiency was 24% and 69%, respectively. The interference efficiency at 48 h was the highest. In the interference group, the content of alanine first increased and then decreased, and the content of carnosine first decreased and then increased, reaching the maximum at 48 h. Under low salt stress, the expression of Sc-CARNS mRNA increased, the content of alanine decreased, and the content of carnosine increased after interference for 0–24 h. After interference for 24 h and 96 h, the mRNA expression of Sc-CARNS and the contents of alanine and carnosine did not change significantly in control group and diethyl pyrocarbonate treated water. The expression level of Sc-CARNS mRNA in the interference group first decreased and then increased with the interference time, the content of alanine first increased and then decreased with time, and the content of carnosine first decreased and then increased. At 48 h, the expression of Sc-CARNS mRNA and the content of carnosine were significantly decreased, and the content of alanine was significantly increased. The results showed that S. constricta has a variable osmotic pressure. Ala as a FAA in vivo contributed the most to osmotic regulation. Moreover, carnosine synthetase was the key enzyme for converting alanine to carnosine. This study revealed the key mechanism of osmotic pressure regulation under low salt stress and provided a basis for revealing the unique salinity tolerance mechanism of Solenida shellfish.

Abstract:The structure and function of microbial communities in aquaculture water bodies are important in the aquaculture ecosystem. To comprehensively and systematically evaluate the structure and functional composition of the microbial community in the water during Chinese mitten crab (Eriocheir sinensis) cultivation, the water quality indicators in the crab pond in Chongming District, Shanghai were monitored monthly from June to October 2022. The microbial species and functional structural characteristics in the water environment during the breeding period were analyzed based on metagenomics technology, and their relationships with environmental factors were discussed. The results showed that the major water quality indicators that exceeded the standard in the breeding pond were pH, permanganate index, total phosphorus, and total nitrogen. During the breeding period, the diversity of the microbial community in the water body from June to August and the richness of the microbial community from July to August were high, and the dominant microbial phyla in the water body were mainly Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, Cyanobacteria, and Uroviricota. At the genus level, the top 10 dominant genera regarding abundance were significantly different among multiple groups. For example, the abundances of Microcystis bacteria in July and unclassified_o_Caudovirales viruses in August–October were significantly higher than those in other months. The primary function of microorganisms is metabolism, including energy metabolism, global and overview maps, and amino acid metabolism. Notable differences are present in the functional composition of different months; particularly, the abundance of metabolic pathways in June–July was significantly higher than that in August–October, and the dominant bacteria (Proteobacteria, Actinobacteria, and Bacteroidetes) were major contributors to the aforementioned functions. The influence trends of environmental factors on the structure and functional composition of microbial communities were consistent. Chlorophyll a and pH had the most significant impact on the structure and functional composition of microbial communities, whereas dissolved oxygen and total phosphorus had a slightly weaker impact. The pathogenic bacteria with a relatively large abundance in aquaculture water bodies are Salmonella enterica, Edwardsiella ictaluri, and Staphylococcus aureus. The research results provide basic data for the study of microbial community structure and functional composition in water bodies of Chinese mitten crab breeding ponds and can provide a theoretical basis for aquaculture water quality control and ecosystem construction.

Abstract:Crassostrea gigas, also known as Pacific oysters, are economic shellfish with the widest range of cultivation, the highest yield in the world, and the most important type of mariculture shellfish in China. However, many C. gigas have died during summer in coastal areas worldwide in recent decades. In 2008, the mortality rate of C. gigas cultured in France reached 40%–100%. In 2009, the mortality rate of C. gigas in some area of Sanggou Bay reached 51%. In 2019, the mortality rate of the Rushan area reached 50%–90%, with the death peak occurring in middle and late August. There were many reasons for the large-scale death of C. gigas, such as temperature, dissolved oxygen, salinity, disease, food availability, and reproduction levels, among which high temperature was the most important abiotic stress factor. The high temperature in summer disturbed the enzyme metabolism of C. gigas, resulting in slow or impeded growth. Furthermore, the reproduction and spawning of C. gigas caused a large amount of protein consumption, and physical weakness combined with high-temperature stress induced many deaths. Therefore, considering the problems faced by C. gigas culture during high summer temperatures, the introduction of new varieties will increase the economic benefits to the industry. Due to its high sterility, triploid C. gigas has attributes such as a fast growth rate, resilience excellent economic characteristics, and high energy conversion efficiency. In recent years, a certain farmed scale has formed in China, especially in northern coastal areas. There have been many studies on the biological and physiological differences between triploid and diploid C. gigas worldwide, mainly focusing on the differences in growth characteristics, soft tissue components, gonadal development, disease resistance, and gill structure. However, comparisons between triploid and diploid C. gigas feeding, metabolic physiology, energy budget, and carbon budget have not been reported. Focusing on the specific period of high temperatures in summer, this study investigated the feeding and metabolic physiological characteristics of triploid and diploid C. gigas using the field flow method, and compared and analyzed their energy allocation strategies in response to a high-temperature environment. The study provide data support for revealing the physiological differences caused by the ploidy effect of C. gigas in order to assist with evaluating the culture capacity. Triploid and diploid C. gigas were selected as research objects in August 2022 to analyze the differences in feeding and metabolic physiology and energy/carbon allocation strategies during high temperatures in summer. Physiological parameters related to intake and metabolism, such as water filtration rate, absorption efficiency, oxygen consumption rate, and ammonia discharge rate, were determined based on the field flow method in Sanggou Bay, Rongcheng, Shandong Province, and energy allocation and carbon allocation were estimated based on the principle of the energy budget. The results revealed that the water filtration rate and assimilation efficiency of triploid C. gigas were higher than those of diploid C. gigas, but there were no significant differences (P>0.05). There were significant differences in the oxygen consumption rate and ammonia discharge rate between triploid and diploid C. gigas (P<0.05). The oxygen consumption rate of triploid C. gigas was significantly lower than that of diploid C. gigas (P<0.05), but ammonia discharge rate was significantly higher than that of diploid C. gigas (P<0.01). The results of the energy and carbon budget analyses showed that the feeding energy/carbon and assimilation energy/carbon values of triploid C. gigas were higher than those of diploid C. gigas, but there was no significant difference (P>0.05). There were significant differences in respiratory energy/carbon, excretion energy/carbon, and growth power between triploid and diploid C. gigas (P<0.05). Respiratory energy/carbon values of triploid C. gigas were significantly lower than those of diploid C. gigas (P<0.05), but excretion energy/carbon and growth power values were significantly higher than those of diploid C. gigas (P<0.05). The oxygen/nitrogen ratio of triploid and diploid C. gigas fluctuated in the range of 7.91–14.11 and 59.81–94.19, respectively. Moreover, the main energy supply substances of triploid C. gigas were proteins, while the main energy supply substances of diploid C. gigas were carbohydrates and fats. These results revealed the differences in energy allocation patterns associated with the ploidy effect of C. gigas during high temperatures in summer. From the perspective of individual physiology and ecology, this study found that, compared with diploid C. gigas, triploid C. gigas showed certain advantages in energy allocation strategies by adjusting feeding and metabolic physiological behaviors during the high-temperature summer. However, the internal molecular mechanism of response strategies adopted by triploid C. gigas to cope with an adverse environment is still unclear. Further interpretation at the molecular level needs to be combined with omics and other systems biology techniques.

Abstract:Penaeus vannamei is an important shrimp species that is farmed globally, owing to its fast growth and good environmental adaptability. However, vibriosis outbreaks, such as acute hepatopancreatic necrosis disease and translucent post-larva disease, have become a considerable constraint on the development of the shrimp aquaculture industry globally in recent years. Due to the negative consequences associated with the use of antibiotics to control bacterial diseases, alternative technologies, such as improving shrimp immunity, the nutrition level of shrimp diets, and the ecological environment of shrimp ponds by using probiotics, have been developed. Owing to the limitations related to the sourcing and cost of fish meal, a large amount of vegetable protein is often used as a fish meal substitute in shrimp artificial feed, which is not only difficult for the shrimp to digest and absorb but can also lead to nutritional antagonism. In order to improve the health status of aquaculture animals by providing high-quality feed, the development and optimization of fermented feed have received extensive attention. Probiotics in fermented feed can produce exogenous cellulase, protease, amylase, and other digestive enzymes in the digestive tract of farmed animals, which can improve the feed utilization rate, promote growth performance, and enhance the immunity of shrimp. In this study, in order to explore the application effects of shrimp feed fermented by different probiotic bacteria, probiotic strains, including Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus acidophilus, were used individually and in combination for the fermentation of commercial feed and fed to shrimp (P. vannamei) for 28 days. The survival, body length growth, specific growth, and feed conversion rates of the shrimp were analyzed, and the density of Vibrio in vitro and in vivo, as well as relevant immune indicators in the serum, were analyzed. Furthermore, ammonia nitrogen and nitrite nitrogen concentrations in the water column were compared between the groups. The results demonstrated that the survival rates of shrimp in the fermented feed groups of B. subtilis, L. acidophilus, and complex bacteria were 8.54%, 8.54%, and 9.76%, respectively, which were significantly higher than that of the control group (P<0.05). The body length growth rates in the B. subtilis and L. acidophilus groups were significantly higher than that of the control group (P<0.05), while the feed conversion rate in the L. acidophilus group was significantly decreased compared with that of the control group (P<0.05). There was no significant difference in the specific growth rate of shrimp between the experimental groups and the control group (P>0.05). Fermented feed can affect the composition of bacteria in the hepatopancreas of shrimp. The density of Vibrio in the hepatopancreas of shrimp in each experimental group was significantly lower than that in the control group on days 14 and 21 of the experiment (P<0.05). The feeding of different fermented feed in the short term (within 14 days) had no significant effect on reducing the total density of Vibrio in the water. However, feeding B. subtilis and L. acidophilus fermented feed significantly reduced the Vibrio density in the water body on day 21 (P<0.05). In addition, the serum total protein concentration in each experimental group was significantly higher than that in the control group (P<0.05). The activities of serum peroxidase, superoxide dismutase, and phenoloxidase in the L. acidophilus fermented feed group increased significantly compared with those of the other experimental and control groups (P<0.05). The activities of serum peroxidase and phenoloxidase in the serum of shrimp fed S. cerevisiae fermented feed were significantly higher than those of the control group (P<0.05). Complex bacteria-fermented feed could significantly increase the activities of serum lysozyme, peroxidase, and phenoloxidase (P<0.05). However, the activity of the detected immune-related enzymes in the B. subtilis group did not increase significantly compared with that of the control group (P>0.05), except for the activity of total serum protein, which was higher than that of the control group. The results also revealed that the concentration of ammonia nitrogen and nitrite nitrogen in the water group of each experimental group at the end of the experiment was significantly lower compared to that of the control group. S. cerevisiae and L. acidophilus groups had the best effect in reducing the concentration of ammonia nitrogen and nitrite nitrogen in the water, respectively. In summary, shrimp feed fermented by B. subtilis, S. cerevisiae, and L. acidophilus singly or in combination could improve the survival rate, growth performance, and immunity of shrimp to a degree, and reduce the concentration of ammonia nitrogen and nitrite nitrogen in aquaculture water. Moreover, L. acidophilus shows great potential as a candidate probiotic for shrimp feed fermentation. The results of this study provide a theoretical basis for the development of shrimp fermentation feed, which can improve the feed utilization and survival rates of shrimp in aquaculture.

Abstract:Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasitic microsporidian species that infects the hepatopancreas of shrimp and causes hepatopancreatic microsporidiosis (HPM). It was discovered in stunted Penaeus monodon in Thailand in 2004. It is a fungal pathogen that can infect various crustacean hosts. It affects farmed shrimp in Southeast Asia and South America, including Thailand, Vietnam, China, Indonesia, India, Malaysia, South Korea and Venezuela. An E. hepatopenaei infection does not cause death in shrimp, but can lead to necrosis and rupture of the hepatopancreatic epithelial cells. This results in reduced digestive and absorptive functions of the hepatopancreatic gland and impaired nutrient storage functions. Therefore, an EHP infection usually leads to shrimp growth retardation or stagnation that greatly reduces shrimp production and seriously affects the high-quality development of shrimp farming. E. hepatopenaei infections were detected in farmed shrimp in coastal provinces of China since 2013 and their high infection rate causes serious economic losses to the cultured shrimp industry. It is difficult to confirm EHP infections solely based on the symptoms of diseased shrimp in the field owing to the extremely small size of EHP and the lack of obvious symptoms in the early stages of EHP infection. Furthermore, the early stages of infection are difficult to accurately detect by light microscopy and histopathological sections. Pathological changes of EHP infection are only observed in the middle and later stages by histopathological sections. In addition, tissue section preparation is time- consuming and complex. This makes it unsuitable as a practical technical method to detect EHP. In contrast, molecular biology methods have become a common laboratory method to detect EHP. This study collected 936 shrimp samples from coastal areas in China from 2021 to 2022, detected EHP positive samples by TaqMan quantitative polymerase chain reaction (qPCR), and analyzed the morphology by histopathology. This study aims to clarify the prevalence of EHP in major shrimp farming species in coastal provinces and cities in China from 2021 to 2022, and to clarify the hazard risk of EHP in cultured shrimp. TaqMan qPCR assays showed that the positive detection rate of EHP in shrimp samples from coastal areas was 10.67% (54/506) in 2021. There was a decreasing trend compared with the national aquatic animal disease surveillance plan from 2017 to 2020. However, the positive rate of EHP in shrimp samples from coastal areas reached 13.72% (59/430) in 2022. There was an increase in the EHP positive detection rate compared with the national surveillance data from 2019 to 2020. The detection results of EHP in samples collected from different regions showed that the positive rate of EHP was high in samples collected from Liaoning (10%) and Shandong (18.80%) in 2021, and in samples collected from Liaoning (14.63%), Hebei (29.17%), Tianjin (28.57%), and Shandong (16.88%) in 2022. The results of samples collected from different shrimp varieties showed that EHP was mainly detected in P. vannamei in 2021 and 2022, with positive rates of 14.10% (54/383) and 16.71% (58/347), respectively. Among them, one sample of Exopalaemon carinicauda was positive for EHP, while no EHP positive sample was detected in Macrobrachium rosenbergii, Protocrayfish cruzi, Penaeus monodon, Marsupenaeus japonicus, and Penaeus chinensis. This study collected and analyzed 506 and 430 shrimp samples from coastal provinces and cities in China in 2021 and 2022, respectively by TaqMan qPCR. These detection results are more reliable than those of previous studies owing to the large sample size and the high specificity and sensitivity of the detection method. The results provided an important reference for a comprehensive understanding of the epidemic situation of EHP in major cultured shrimp in coastal areas of China from 2021 to 2022. Histopathological examination of EHP-positive P. vannamei revealed that scattered or clustered EHP spores and EHP protoplasts were observed in hepatopancreatic epithelial cells. The epidemiological survey and molecular epidemiological analysis of shrimp farmed in coastal provinces, municipalities, and some inland provinces of China in 2021 to 2022 showed that the prevalence rate of EHP is generally decreasing compared to previous years. This indicated that remarkable achievements were made to prevent and control EHP in China's shrimp farming industry in recent years. However, EHP is widely prevalent in farmed shrimp in coastal provinces in Northern China. Therefore, measures such as strict EHP quarantine of brood stock and seedlings and the promotion of non-EHP seedlings should be taken to further reduce the epidemic range and risk of EHP and promote the green and high-quality development of the shrimp culture industry in China.

Abstract:Acute hepatopancreatic necrosis (AHPND) is a bacterial disease caused by Vibrio bacteria that severely affects the Pacific white shrimp farming industry. Since AHPND was first identified in China and Vietnam in 2010, it was subsequently identified in Malaysia (2011), Thailand (2012), Mexico (2013), the Philippines (2014), South Korea (2016), Bangladesh (2017), the United States (US) (2017), and Japan (2020). AHPND causes significant economic losses of more than 7 billion US$ annually to the global shrimp farming industry. It has severely restricted the development of the global shrimp farming industry. Many studies have shown that the pathogens of AHPND are Vibrio spp., including V. parahaemolyticus, V. owensii, V. campbellii, V. harveyi, V. punensis, and V. anguillarum. The virulence of these pathogenic strains was derived from a 70 kb virulent plasmid (pVA1-type plasmid). The pVA1-type plasmid carries pirAB genes encoding the binary toxin pirAB homologous to the insecticidal protein of Photobacterium phosphoreum. The pVA1-type plasmid has been confirmed to carry a novel trb type Ⅳ secretion system (T4SS). Trb-T4SS can mediate the conjugative transfer of the pVA1-type plasmid at high cell density for interspecific horizontal transfer. The quorum sensing (QS) system is also known as the density-sensing system. In the surrounding environment, the QS system allows bacteria to regulate the expression of multiple genes by sensing changes in the concentration of autoinducer signaling molecules. At high cell density, high concentrations of autoinducer signaling molecules bind to receptor proteins to inhibit phosphorylation cascades and high cell density master regulator OpaR is normally expressed. OpaR is involved in the regulation of various biological processes, such as biofilm-forming ability, motility, and the expression of the type Ⅲ and type Ⅳ secretion systems. Reportedly, high concentrations of signaling molecules in the QS system regulate the expression of key genes of the T4SS to increase the conjugative transfer efficiency of drug-resistant plasmids. However, the regulation of the T4SS by opaR in AHPND is not yet reported. In this study, OpaR, the high cell density master regulator of the QS system in V. parahaemolyticus, was selected to explore the relationship between the QS system and T4SS expression, as well as the conjugative transfer of the pVA1-type plasmid. The V. parahaemolyticus 20130629002S01::cat (Vp2S01::cat) strain, a pathogenic strain of AHPND, was used as the starting strain. The opaR gene was replaced with the erythromycin resistance gene (ermB) by homologous recombination and electroporation. The opaR mutant strain (Vp2S01::catΔopaR) was successfully constructed. The effects of OpaR on Vp2S01::cat were explored by performing growth curve and motility assays. Vp2S01::cat and Vp2S01::catΔopaR were cultured continuously for approximately 24 h in a shaking bed at 28 ℃ and 180 r/min. OD600 was measured every 2 h to compare the difference in growth. The results showed that there were no significant differences in the growth curves for Vp2S01::cat and Vp2S01::catΔopaR. Biofilm-forming ability was detected using a crystal violet staining assay. The results showed that the biofilm-forming ability of Vp2S01::catΔopaR was significantly reduced. Growth zone diameter was recorded in 0.3% (swimming) LB agar plates at 28 ℃ for 8 h to analyze the difference in swimming ability. We found that the swimming ability of Vp2S01::catΔopaR increased significantly by 2.67 times compared with that of Vp2S01::cat. Growth zone diameter was recorded in 1.5% (swarming) LB agar plates at 28 ℃ for 12 h to analyze the difference in swarming ability. The results showed that there was no significant difference in swarming ability between Vp2S01::cat and Vp2S01::catΔopaR. However, Vp2S01::catΔopaR had more missing colonies. Using VcLMB29 as the receptor strain, the conjugative transfer efficiency of Vp2S01::cat and Vp2S01::catΔopaR was compared at different time points. We found that the conjugative transfer efficiency of Vp2S01::catΔopaR increased after 12 and 24 h. The conjugative transfer efficiency after 24 h was increased by a factor of 265.43. RNA of Vp2S01::cat and Vp2S01::catΔopaR was extracted after 24 h of conjugative transfer and reverse-transcribed into cDNA for real-time quantitative fluorescence PCR (RT-qPCR). GyrB was used as the reference gene. AphA-qRT, opaR-qRT, and T4SS-qRT primers were used for RT-qPCR. The relative expression levels of genes were calculated using the 2–ΔΔCt method. The results showed that the relative expression levels of T4SS in the Vp2S01::catΔopaR experimental group were significantly increased by 1.13–3.21 times and that the relative expression levels of the key conjugative transfer genes traF, trbE, and traG were significantly increased by 1.96, 1.92, and 3.21 times, respectively. In conclusion, the opaR gene does not affect the growth and swarming motility but does affect the biofilm-forming and swimming ability of Vp2S01::cat. The high cell density master regulator OpaR may affect the conjugative transfer efficiency of the virulent plasmid by regulating the expression levels of T4SS. This study provides fundamental data for analyzing the mechanism by which the QS system regulates the expression of T4SS and the conjugative transfer of the pVA1-type plasmid in AHPND pathogenic bacteria, offering technical support for the control of the horizontal transfer of the virulent plasmid in AHPND-causing bacteria.

Abstract:This study is committed to developing and optimizing a rapid nucleic acid preparation reagent (nucleic acid release agent) that is suitable for recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) technology to detect DNA nucleic acid samples in shrimp hepatopancreas to realize on-site rapid detection and remove the cumbersome and time-consuming steps of conventional nucleic acid sample extraction. The optimum compositions of nucleic acid release agent are 20–100 mmol/L Tris-HCl, 50–250 mmol/L KCl, 0.01%–0.10% (W/V) lithium dodecyl sulfate (LDS), 0.5%–2.0% (V/V) Triton X-100, 1–5 mmol/L ethylenediaminetetraacetic acid disodium salt (EGTA2Na), 0.5–5.0 mmol/L bovine serum albumin (BSA), 1–5 mg/ml gelatin, 0.01%–0.10% (W/V) trehalose, and 1%–5% (W/V) betaine. The proportion of each component of nucleic acid release agent was optimized using positive and negative samples of shrimp Enterocytozoon hepatopenaei (EHP). The RPA-LFD reaction initially involved collecting shrimp hepatopancreas (the size of a mung bean), adding 100 μL nucleic acid release agent, heating for 3 min at 100 ℃ and using the supernatant for analysis. The optimal ratio of each component of nucleic acid releaser was determined to be 100 mmol/L Tris-HCl, 100 mmol/L KCl, 0.02% LDS, 0.5% Triton X-100, 1 mmol/L EGTA2Na, 0.05% trehalose, 1 mg/mL gelatin, 0.5 mmol/L BSA, and 2% betaine. The positive and negative samples of acute hepatopancreatic necrosis disease (AHPND) were used as detection templates to verify the optimized nucleic acid release agent. The optimized nucleic acid release agent could be used to prepare nucleic acid samples, and the prepared samples were available for RPA-LFD method testing. The optimized nucleic acid release agent in this study can be used to prepare DNA nucleic acid samples of the shrimp hepatopancreas for RPA-LFD detection. It avoids the tedious and time-consuming preparation steps of conventional DNA nucleic acid samples and greatly improves the efficiency of nucleic acid level pathogen detection. Disease is the bottleneck that restricts the green development of the shrimp breeding industry, which can cause huge annual economic losses. Detection and monitoring of shrimp related pathogens are effective to solve this problem. Enterocytozoon hepatopopenaei (EHP) and Vibrio parahaemolyticus (VP) are two common pathogenic microorganisms in shrimp culturing that cause AHPND. E. hepatopopenaei is an obligate intracellular parasite that mainly parasitizes in the hepatopancreas of shrimp. Severe EHP infections lead to atrophy of the hepatopancreas of shrimp. E. hepatopopenaei has a wide range of hosts, including Penaeus vannamei, P. monodon, and Palaemon carincauda. AHPND is an aquatic disease caused by Vibrio parahemolvticus with the virulent plasmid PirA/B. It was first found in China and Vietnam in 2010, then in Malaysia, Thailand, Mexico, the Philippines, and other countries. P. monodon, P. vannamei, P. chinensis, and P. japonicus are susceptible hosts. The distribution of pathogenic vibrio can be detected in the hepatopancreas tissue of diseased shrimp, and it can cause hepatopancreas atrophy. Broadly, most shrimp pathogens (such as DIV1, WSSV, TSV, and IHHNV) infect the hepatopancreas of shrimp. DIV1 infections cause pale atrophy of the hepatopancreas. Histopathological examination identified eosinophils and dark eosinophilic inclusions in the cytoplasm of the hepatopancreas of shrimp infected with DIV1. Some of them were coated or contained slight basophilic staining, and hemocytes were pyknotic. WSSV and TSV can cause pale atrophy symptoms of the hepatopancreas, while IHHNV can also cause hepatopancreas enlargement of shrimp. Therefore, the nucleic acid samples of the hepatopancreas of shrimp can be widely used as templates for detection of most shrimp pathogens. Isothermal nucleic acid amplification technology removes the dependence on conventional nucleic acid temperature change amplification using the PCR amplification instrument, and is widely used for rapid detection. Currently, isothermal amplification technology covers loop mediated isothermal amplification (LAMP), strand displacement amplification (SDA) and recombinase polymerase amplification (RPA), and so on. Among them, RPA technology was developed in recent years using recombinases to promote the insertion and binding of oligonucleotide primers in DNA double strand complementary sequences, and realize exponential amplification of specific DNA regions under the action of Bsu DNA polymerase. It has a short reaction time, strong specificity, high sensitivity, and operates at a constant temperature of 37–42 ℃. It is suitable for on-site detection and analysis. The combination of RPA with the lateral flow dipstick (LFD) to read the detection results is simple to operate, fast, with high specificity and sensitivity. It is a new method suitable for rapid detection in the field. The combination of RPA-LFD technology and a simplified nucleic acid extraction method can help to realize portable on-site rapid nucleic acid detection.

Abstract:Penaeus vannamei is an important breeding shrimp species in China. Moreover, the quality of the larvae is the cornerstone for the sustainable development of the shrimp industry. In recent years, shrimp postlarva bacterial vitrified syndrome (BVS) has caused huge economic losses in the shrimp postlarva breeding industry, with epidemiological characteristics of rapid onset and high mortality, occurring at 2–3 days post-hatch. The diseased postlarva symptoms include emaciation, decreased activity, and an empty intestinal tract and stomach. The hepatopancreas shows atrophy, blurring of contour, paleness, and even vitrified syndrome. The causative agents of the disease are Vibrio alginolyticus and Vibrio parahaemolyticus. Furthermore, antibiotics are often used to treat BVS; however, frequent and inappropriate use of antibiotics can lead to bacterial resistance and drug residues. With the advantages of antibacterial activity, immunity regulation, low toxicity, few side effects, and lack of drug resistance and drug residues, Chinese herbal medicines play an important role in aquaculture. There are currently no effective drugs available to prevent and treat BVS. It is urgent and important that effective Chinese herbal compounds are developed for BVS prevention and treatment. In this study, first, an antibacterial test was carried out in vitro by the Oxford cup and double dilution methods. Overall, 50 types of Chinese herbal medicines were screened, and those with a good bacteriostasis effect on V. alginolyticus and V. parahaemolyticus were identified. Then, a pathological model of BVS was constructed with different concentrations of V. alginolyticus. The efficacies of the Chinese herb compounds were evaluated based on the mortality, histopathology and ultrastructural pathology characteristics of the postlarvae. The bacterial inhibition test showed that the inhibition zone of V. alginolyticus could be > 12 mm with the application of one of six types of Chinese herbs, including Schisandrae chinensis fructus, Verbenae herba, and Granati pericarpium. The inhibition zone of V. parahaemolyticus could be > 13 mm with the application of one of five types of Chinese herbs, including Chebulae fructus, S. chinensis fructus, Moutan cortex and Sanguisorbae radix. Then, 11 types of antibacterial and two types of immunological drugs were selected for further analysis. Moreover, the MIC and MBC of G. chinensis, C. fructus, Caryophylli flos, and S. chinensis fructus against V. alginolyticus and V. parahaemolyticus were ≤ 12.5 mg/mL and ≤ 50 mg/mL, respectively. Then, three types of compounds were used for in vivo pharmacodynamics tests by combining Chinese antibacterial medicine with Chinese immunological medicine (Cyperi rhizoma and Gardeniae fructus), which were named prescription 1 (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g), prescription 2 (C. fructus 40 g, G. pericarpium 30 g, C. flos 30 g), and prescription 3 (C. fructus 40 g, G. chinensis 30 g, G. fructus 30 g), respectively. Florfenicol was used as the positive drug control, and V. alginolyticus infection without treatment was used as the positive control. The BVS model indicated that V. alginolyticus caused the same vitrification symptoms in shrimp postlarvae as a natural infection. The LD50 of shrimp postlarvae was 2.82 × 105 CFU/mL after 72 h of infection with V. alginolyticus. The pathological model of BVS was constructed by immersing shrimp postlarvae with 5 × 104 CFU/mL V. alginolyticus. Based on this, the infected shrimp postlarvae were treated with drugs for 7 days. After this period, the mortality rates of each group from low to high were: blank control < prescription 1 < florfenicol < prescription 3 < prescription 2 < positive control. The mortality rate of the shrimp postlarvae in the prescription 1 group was significantly lower than that in the positive control group (P < 0.05). The mortality rate of the shrimp postlarvae in the prescription 2 and 3 groups and the florfenicol group was lower than that in the positive control group, but the difference was not significant (P > 0.05). Thus, prescription 1 can significantly reduce the mortality of BVS-positive shrimp postlarvae. Histopathology observation showed that after 7 days of administration, the hepatopancreas and intestinal epithelium of larvae in the prescription 1 group were more intact, with more hepatic tubules, less exfoliation and necrosis of the epithelial cells, and more abundant and well-arranged intestinal epithelial cells. Ultrastructural pathology observation showed that after 7 days of administration, the cell membrane and nucleus of the liver tubule epithelial cells of the diseased shrimp postlarvae in the prescription 1 group were normal, and the mitochondria and endoplasmic reticulum were abundant. Histopathology observation showed that the hepatopancreas and intestinal tract lesions of the shrimp postlarvae in the prescription 1 treatment were significantly less than those in the other groups. In summary, Chinese herbal compounds (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g) had the best effect for preventing and treating shrimp postlarva BVS caused by V. alginolyticus. The results of this study lay a scientific foundation for the development of specialized herbal compounds for the prevention and treatment of bacterial infections and contribute to the green and high-quality development of the shrimp industry.

Abstract:Macrobrachium rosenbergii is one of the most popular species in aquaculture. However, the M. rosenbergii farming industry has been facing an ongoing iron prawn syndrome (IPS) crisis since 2010, resulting in substantial economic losses to the farming industry. Infectious precocity virus (IPV) is a novel virus of Flaviviridae found in recent years that can cause sexual precocity and associated slow growth in healthy M. rosenbergii. It is believed to have a specific correlation with IPS. There are very few cell lines of crustaceans that can be used for studying the response of cells to pathogens. Even though the primary culture technology of blood and muscle cells in M. rosenbergii has gradually matured, there have been few studies on the primary culture of neural cells. Quantitative results of different tissues of IPV-positive M. rosenbergii have indicated that nerve-rich tissues, such as eyestalk, brain, and thoracic ganglion tissues, have a higher viral load, which explains why IPV has neurotropic tissue characteristics. To provide an in vitro cell platform for studying the virus-host interactions of IPV, a simple, stable, and feasible primary culture method was established for the nervous tissue primary cells of M. rosenbergii. In this study, healthy prawns with a body length of about 10–12 cm, healthy appendages, and vitality were selected for the experiments. The body surface of M. rosenbergii was first disinfected with 75% alcohol. On the clean bench, the nervous tissues, including the brain, X organ-sinus gland complex from the eyestalk, thoracic ganglion, and abdominal ganglion tissues, were isolated and washed in PBS buffer containing 100 U/mL penicillin and streptomycin, 100 U/mL amphotericin, and 80 U/mL gentamicin, 2–3 times. The tissues were then placed in 5 mL of 0.5% papain solution. After digestion at 25 ℃ for 5 min, 1× L-15 medium containing 15% FBS, 140 mmol/L D-glucose, and antibiotics (100 U/mL penicillin and streptomycin, 100 U/mL amphotericin, and 80 U/mL gentamicin) were added to terminate digestion. Next, the cells were seeded onto a 24-well plate and allowed to settle in darkness at 25 ℃ for 45 min. After adhesion, cells were transferred to a cell incubator and cultured in the dark at 28 ℃. The nervous tissues’ primary cells at different time points were observed under an inverted microscope, and the morphological changes were recorded through imaging. The compound eye tissue of IPV-positive M. rosenbergii was placed in SM buffer, and the IPV mixture was obtained after multiple rounds of crushing and centrifugation. The brain tissue primary cells with large quantities and better culture effects were selected for the IPV challenge after cultured in vitro for 5 days. The virus crude extract was filtered through a 0.22 μm filter membrane and mixed according to the volume ratio of virus crude extract to serum-free medium = 1∶9. For the experimental group, 2 mL of the mixed solution was added to each well, while the control group received 2 mL of serum-free medium per well. Samples were taken at 0, 6, 12, 24, 48, and 96 h after virus infection. RT-qPCR was used to detect the IPV load. The results showed that the cultured primary cells grew well in a 1× L-15 medium containing glutamine and serum. According to the morphology of the cells, number of axons, and other characteristics, brain tissue primary cells, primary cells from the eyestalk X organ-sinus gland complex, and thoracic ganglion tissue primary cells were divided into four types: neurosecretory cells, pseudounipolar neural cell-like, bipolar neural cell-like, and multipolar neural cell-like, respectively. In comparison, only round neural cells and bipolar-like neural cells were found in the primary cell culture of the abdominal ganglion tissue under in vitro culture. The brain tissue primary cells and X organ-sinus gland complex cells survived for 15 days, while the thoracic ganglion and abdominal ganglion tissue primary cells survived for 9 days after being cultured in vitro. After IPV infected the brain tissue primary cells, the viral load of IPV was 15.30 copies/μg RNA at 6 h post-infection (hpi) and 35.59 copies/μg RNA at 12 hpi. IPV was not detected in IPV-infected brain tissue primary cells at 24 hpi and 48 hpi. Then, IPV viral load was detected at 96 hpi, reaching 104 copies/μg RNA. In conclusion, this study successfully established a simple and convenient primary culture technology for cells from M. rosenbergii neural tissues, providing preliminary data and a platform for neuroendocrine and virus-host interactions research. An in vitro infection model of IPV was also initially established in this study. Before establishing the shrimp cell line, the cells could be used to study the mechanism of viral infection, replication, and transcription, providing primary data and a platform for studying the norovirus and neuroendocrine factors of M. rosenbergii. They could also provide critical experimental materials for further research into infection mechanisms and the development of virus and disease prevention technologies.

Abstract:Paralytic shellfish toxins (PSTs) are a class of acute neurotoxins with nearly 60 congeners. Shellfish that filter feed on toxin-producing algae exhibit PST accumulation in tissues, and the consumption of toxic shellfish poses a major threat to human health. China has the largest aquaculture industry in the world, with shellfish production of approximately 15.89 million tons in 2022 and many shellfish consumers. The PST poisoning incidents in China exhibit the characteristics of a wide distribution area, recurring regional risk, diverse toxin sources, and seasonal differences. PST consumption causes acute symptoms of poisoning and even death in many people, with a high lethality rate. This is a serious threat to the health of consumers and detrimental to the social stability of the region. In northern China, Hebei Province has rich fisheries resources, with a mariculture output of approximately 580,000 tons in 2022. In recent years, PSTs have been detected several times in bivalve shellfish inshore in Hebei Province, especially along the coast of Qinhuangdao. A serious shellfish poisoning episode was reported in Qinhuangdao in 2016, with PST concentrations exceeding the safety limit of 800 μgSTX equivalents/kg (μg STXeq/kg) by a factor of 65. The main algal species responsible for the poisoning was Alexandrium catenella. However, the current investigations of PST pollution in Hebei Province mainly focus on the shellfish species Mytilus galloprovincialis and Argopecten irradians in the sea area of Shanhaiguan, Qinhuangdao, and investigations on other coastal cities of Hebei Province and a wide range of different shellfish species are lacking. Moreover, few risk assessment studies have been conducted. A continuous PST survey was conducted in 2022 on six species of shellfish, namely M. galloprovincialis, Scapharca subcrenata, A. irradians, Scapharca broughtonii, Ruditapes philippinarum, and Crassostrea gigas, collected by netting in Tangshan and Qinhuangdao, and the residual status of PSTs was assessed for acute exposure using liquid chromatography-tandem mass spectrometry. The results showed that PSTs were detected in shellfish samples collected from March to June, with a detection rate of 100%. However, no values exceeded the European Union limit. Among the shellfish samples collected from March to June, those assessed in April had the highest average concentration, followed by those in May, March, and June, which had the lowest average concentration. The main components detected in shellfish samples from March to June were gonyautoxins (GTX) 1–4; the highest concentrations were detected in April. Highly toxic saxitoxin (STX) and decarbamoylneosaxitoxin (dcNEO) were detected from April to June and not in March. Decarbamoylgonyautoxin (dcGTX) 2 was not detected in March or April; however, it was detected in May and June. The sea area near Qinhuangdao was more polluted than that near Tangshan. Among the collected shellfish samples, significant differences were observed in the average concentration of PSTs in shellfish samples of different species, and the concentration ranged from highest to lowest in species as follows: M. galloprovincialis > S. subcrenata > A. irradians > S. broughtonii > R. philippinarum > C. gigas. Large differences were observed between the six shellfish species regarding the accumulation of 11 PST components, among which GTX1–4 and carbamatetoxin (C) 1 and 2 were the most commonly detected components. The highest concentrations of GTX1 and 4 were observed in M. galloprovincialis, the highest concentrations of GTX2 and 3 were observed in S. subcrenata, and the highest concentrations of C1 and 2 were observed in A. irradians. A low detection rate was recorded for highly toxic STX and neosaxitoxin (NEO in samples, except in M. galloprovincialis and S. subcrenata, in which all 11 PSTs were detected. In the other four shellfish species, only certain of the components were detected. Statistical analysis of the highest levels of PSTs in nearshore shellfish in Hebei Province in recent years showed a decreasing trend. Acute exposure assessments using the maximum value of PSTs showed that none of the six shellfish species exceeded the acute reference dose values recommended by the Food and Agriculture Organization of the United Nations/World Health Organization and European Food Safety Authority, suggesting that toxin levels were safe and acceptable. M. galloprovincialis had the highest dietary exposure risk, as a few residues were present, and symptoms of PST poisoning, such as dizziness and nausea, may occur after ingesting large quantities of M. galloprovincialis. Increased attention should be paid to shellfish with high dietary exposure assessment values, such as M. galloprovincialis and S. subcrenata. In this study, we investigated PSTs in shellfish from Hebei coastal waters and observed that PSTs were detected in shellfish samples from March to June, with the highest average concentration in April, followed by May and March, with the lowest average concentration in June. In addition, the main components detected in shellfish samples were GTX1–4. The analysis of different shellfish species showed that M. galloprovincialis had the highest average PST concentration, followed by S. subcrenata, A. irradians, S. broughtonii, R. philippinarum, and C. gigas; GTX1–4 and C1 and 2 were detected in all six shellfish species. Dietary exposure assessment for consumers in Hebei Province showed that the toxin levels were safe and acceptable. However, follow-up surveys and studies are necessary due to the paucity of surveys for different cities and species on the coast of Hebei.

Abstract:In the context of global climate change, one central interest is an improved understanding of the global carbon cycle. A large number of studies have investigated carbon cycling and associated elements, mainly nitrogen and phosphorus. However, as an essential element for diatom growth, Si has been largely ignored. Si is the second most abundant element and is widely distributed on Earth. The chemical weathering of silicates on land and photosynthesis of diatoms in the ocean play an important role in atmospheric CO2 levels at various timescales. Diatoms are the primary producers in the ocean and account for as much as 40% of the annual ocean carbon fixation, which have an absolute requirement for Si to form siliceous cells. The main mechanism underlying ocean carbon sinks is a “biological pump.” The biological pump is driven by the biological Si pump to a large extent. Therefore, the biogeochemical process of Si has become one of the key research issues for global environmental change. Based on previous studies, the regulation and influence of the Si biogeochemical cycle on the carbon cycle are discussed in this review. The coupling effect and mechanism of the Si and carbon cycles in shellfish culture ecosystems were analyzed and the key research questions were explored. Chemical weathering of silicates and the cycling of their products form the basis of Si biogeochemistry. CO2 is consumed during weathering reactions. Therefore, silicate weathering on land represents an important sink for atmospheric CO2. Furthermore, at the geological timescale, primary silicate mineral weathering is the source of secondary silicate. Terrestrial plants absorb soluble silica through their root system during growth. Amorphous silica deposited in plant tissue after maturity is called phytolith. Phytoliths have excellent geochemical stability and occlude a certain amount of organic carbon during the formation process. The organic carbon occluded within phytolith is called phytolith-occluded carbon (PhytOC) and is buried in the soil. PhytOC is released into the soil with phytolith and may be preserved in soils for several thousands of years. As a consequence, PhytOC in terrestrial ecosystems could be significant potential carbon sinks globally due to the refractory phytolith. Primarily through river input, the dissolved silicate (DSi) is transported into the coastal ocean (approximately 84% of DSi input to the oceans). As the major primary producer, diatoms absorb DSi during growth and account for a large fraction of the total carbon fixation in the modern oceans. DSi is converted into biogenic silica via biological processes, is transported to the deep ocean, and is finally buried into sediments with organic carbon in the marine ecosystem. Thus, by controlling the contribution of diatoms to the total primary production, DSi can affect the carbon cycle in oceans. The carbon pump is driven by the Si pump. Mariculture has developed quickly in recent decades. Shellfish, which are dominated by filter-feeding species, are the main mariculture species. The filter-feeding shellfish consume particulate organic carbon as phytoplankton and use dissolved inorganic carbon to build their shell during growth. Filter-feeding shellfish are an import fishery carbon sink. As one of the important feed sources of filter-feeding shellfish, diatoms form fishery carbon sinks in coastal shellfish culture areas. Silicate is an essential salt for diatom growth. Consequently, the carbon sink of filter-feeding shellfish culture is connected with DSi through diatoms. Si could play an important role in driving the formation of carbon sinks in filter-feeding shellfish culture. Hence, it is necessary to consider all processes and coupling effects in the study of the Si biogeochemical cycle. It is important to understand its role in the carbon sinks of shellfish culture. Nowadays, in many systems, human perturbation has resulted in a decline in the ratio of Si:N to 1:1 or less, with severe impacts on the quality and structure of aquatic ecosystems. DSi limitation has been reported in many studies, in both coastal and marine waters. DSi limitation causes shifts from diatoms to non-siliceous algae and is supposedly related to the decreasing export of carbon. A shift from diatoms to other species would enhance the recycling of organic matter in the upper water column because diatoms are very effective in carbon sequestration. DSi limitation has also appeared in some aquaculture bays in China, such as Jiaozhou Bay and Laizhou Bay, in spring. Regarding future directions, it is suggested that more research be conducted on Si biogeochemistry in shellfish culture systems and coupling with the carbon cycle. The subsequent results could evaluate the role of Si in the carbon sink of filter-feeding shellfish culture. Future studies are expected to provide ideas for alleviating Si deficiency in the aquaculture bay and exploring the expansion path in shellfish farming.

Abstract:With the increase in global population, the demand for aquatic products rises annually. As a sustainable industry, aquaculture offers a promising solution to address the growing demand for fish products. Meanwhile, intensifying and expanding sustainable aquaculture is essential for achieving the United Nations’ global goal of the Decade of Action. Cage culture is a typical aquaculture method used globally. This culture method has the following advantages: (Ⅰ) Highly researched with a long history: cage culture has been in use since the 1970s. Cultivating high-value aquatic products through cage culture has become indispensable in aquaculture. (Ⅱ) The development prospects of cage culture are broad and include realization of the comprehensive breeding of different species. In the integrated multi-nutritive aquaculture (IMTA) system, cage culture enables a multi-species combination in which uneaten feed and nutrients from the excreted waste of one breeding species are used as food for other breeding species, thereby reducing nutrients released into the environment and increasing overall productivity. Net cage culture in shallow waters has grown exponentially in the past few decades. However, due to the limited space in nearshore areas and an increasing number of countries paying attention to the environmental problems caused by cage culture, the method has gradually shifted to deeper waters to ensure food security and safety. At the same time, alleviating environmental stress caused by nearshore cage culture and the expansion of the food production space are inevitable. Compared with nearshore aquaculture, the environmental conditions of offshore aquaculture are more complex, and the net cages are inevitably subjected to harsh sea conditions. To achieve the safe production of offshore aquaculture, it is crucial to study wind and wave resistance technology for use in culture net cages. As an essential part of net cages, the netting system is mainly used to maintain breeding space, protect the breeding species, prevent them from escaping, and protect them from predators. In the flow and wave field, the netting system bears most of the load on the farming facilities. Compared with other traditional marine structures, the nets have the mechanical characteristics of small scale and high flexibility. Under external force, the nets show large displacement and massive deformation, reducing breeding space and increasing the likelihood of damage to breeding species due to crowding. At the same time, the nets change the flow and wave field around the facility, affecting the distribution of the remaining bait, breeding species’ excrement, and dissolved oxygen in the water. Currently, the lack of analysis technology for nets is a limitation in offshore cage culture engineering, which restricts the large-scale development of offshore cages. Therefore, research on the hydrodynamic characteristics of nets is vital for developing offshore cage culture. This study introduced primary methods for calculating the netting hydrodynamic loads and their applicability. Simultaneously, the predominant modeling techniques in the numerical calculation of the netting dynamic response were summarized and analyzed. Furthermore, a systematic review of studies relating to the wave flow field around net mesh was conducted. Finally, the current hot topics in the research of netting hydrodynamic characteristics were reviewed to provide a reference for designing and optimizing cages. Considering the influence of various parameters on the hydrodynamic loads on the nets is an effective way to improve the prediction accuracy for the loads, which is also a critical area of research that requires further investment. In order to restore the real force characteristics of nets, a database of the netting hydrodynamic coefficients should be established through experiments. Intelligent algorithms, such as digital twin technology, are used to construct prediction models and generate mapping relationships between multiple factors and the hydrodynamic coefficients of the nets. Moreover, databases and algorithms should be updated regularly to improve the accuracy of calculating the netting hydrodynamic load. For the dynamic response of the nets, the fluid-structure coupling of the flexible nets should be studied intensively in the future, focusing on the two-way coupling between the nets and the fluid and exploring direct numerical simulation methods. Meanwhile, considering computational accuracy, suitable algorithms, such as the submerged boundary method, should be selected, and parallel computational methods should be developed to improve computational efficiency. In terms of the flow and wave field around the nets, further research should be conducted to determine the effects of extreme waves, biofouling, fouling density, cultured fish species, fish size, fish number, fish swimming speed, and fish swimming status on the flow field in the net cages, which will be conducive to monitoring the health of fish, reducing the risk of fish diseases, and ensuring the sustainable development of cage culture. This study provides a reference for developing net hydrodynamic analysis for digitalization and precision. It also provides more information for the sustainable development of aquaculture.