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Abstract:The waters of the northern Shandong Peninsula are spawning and nursery grounds for many fishery organisms from the Bohai Sea and Yellow Sea and play an important supporting role in the supplementation and reproduction of fishery resources in these seas. In recent years, with global warming and intensification of human activities, the fishery resources in Shandong offshore have generally declined, with obvious species miniaturization, low quality, and reduction of species diversity. Important fishery resources have been unable to achieve the minimum amount for the fishing seasons, and its support function in the Bohai and Yellow seas is increasingly diminishing. The fishery organisms research in the northern part of the Shandong Peninsula is less focused on their composition, community structure, and fishery biological health evaluation. Therefore, based on the northern Shandong Peninsula fishery resource survey data of May–June 2021, this study analyzed the fishery's biological diversity and identified the dominant species in these waters. Key species were evaluated through food web topology and social network analysis, and the fishery's biological health status was provided based on the survey data of 2011 and 2013. The study showed that the fishery resources of the Shandong Peninsula would be conserved and managed in 2021. The proportion of pelagic fish in spring decreased in 2021, and the turnover of dominant species showed a gradual decrease in pelagic fish, mainly small and less economically valuable. In the past 40 years, the spring fish diversity and evenness indices in the northern Shandong Peninsula sea area have been continuously reduced, reaching values lower than the reasonable range of the proposed diversity index, which indicates that the species in this area are poorly homogeneous concerning their horizontal structure. The higher number and extremely uneven distribution of species can lead to a lower diversity index. Compared with the late 20th century to the early 21st century, Engraulis japonicus was a dominant resource based on the results of this study, and the lower number of other fishes may be a factor for the lower diversity index. The significant increase in the richness index may be related to the occurrence of succession in the community structure, resulting from the mutual replacement of small, low-quality species with short life cycles, rapid resources renewal, and small individuals. The dominant species E. japonicus is often found among the catch. Shandong offshore fisheries development history corresponds to a significant number of large, high-value class invertebrates fishing, including cephalopods such as Loligo chinensis, in an unhealthy state. Crangon affinis is the absolute dominant species of invertebrates. In the spring of 2021, the fish food web in the northern Shandong Peninsula showed correlation values ranging from 0.03 to 0.30, excluding a possible specific relationship between the population feeding and the community. Moreover, external factors may disturb the community, which is consistent with the community interspecific feeding relationship under natural conditions. The key community species were Liparis tanakae, Lophius litulon, E. japonicus, Larimichthys polyactis, and Chaeturichthys stigmatias. E. japonicus was both the dominant and key species in the fish community in the northern Shandong Peninsula waters in 2021. The regulating effect of E. japonicus on the food web was related to its upward control effect through changes in resource abundance that affects the changes of other fish that use this area as bait. L. tanakae, L. litulon, and L. polyactis were important predators in the sea through the downward control effect, affecting the stability of the whole food web. Fishery biological density, fish density, and crustacean density health index were found within healthy parameters, while the cephalopod density health index was in a sub-healthy state. Compared with 2011–2013, the densities of various economic species have increased significantly, and the overall health of fishery organisms in the northern part of the Shandong Peninsula is good. Overall, the results of this study can provide a reference for conserving and managing fishery resources in the Shandong offshore region.

Abstract:This study used Tweedie-GAM to evaluate the distribution characteristics of Japanese scad (Decapterus maruadsi) in spring, summer, and autumn (from 2015 to 2020) and its relationship with marine environmental factors. The fishery resources data from the southern Zhejiang coastal waters were combined with the hydrological environment survey data collected simultaneously. The results showed significant differences in the Japanese scad spatial distribution in different seasons. Japanese scad is mainly concentrated in the nearshore waters in spring and concentrated primarily on the south and north regions of the Zhejiang southern waters in summer, showing a "high on both sides, low in the middle" pattern. The distribution pattern in autumn is opposite to that in spring, wherein the Japanese scad mainly concentrated in open waters. There are some differences in the key influencing factors of Japanese scad in different seasons. In spring, with a range of 19.5~25.0 ℃, the resource density firstly showed an increasing trend and then decreased with the rise in water temperature, peaking at 22.7 ℃. During summer, the water temperature reached 28.0~30.3 ℃, and the resource density showed the maximum value when the salinity was 32.3. In autumn, the resource density showed a decreasing trend and later increased with the increment in the water salinity, with a minimum value of 29.8. The relationship between resource density and water depth remained stable, showing a multi-wavy nonlinear relationship, with an overall increasing trend and later stable pattern. This study revealed the relationship between the Japanese scad's temporal and spatial distribution characteristics and environmental factors. It provides a research reference for the conservation, management, and sustainable utilization of Japanese scad in the coastal waters of southern Zhejiang.

Abstract:In China, aquaculture is the primary source of aquatic products due to the decrease in wild fishery resources. In 2018, the total output of aquatic products in China expanded to 47.6 million tons, accounting for 58% of global aquaculture production. Intensive culture methods generally use significant quantities of feed however, approximately 75% of nitrogen in the feed is retained in aquaculture water, mainly as soluble nitrogen, such as ammonia nitrogen (NH4+-N) and nitrate (NO3–-N), owing to low feed-utilization rates during cultivation. At the same time, fishes generate a substantial amount of excreta, which will cause the increase of nitrogen compounds in water and negatively affects the quality of aquatic products. Serious problems could occur if nitrogen compounds are discharged into the environment, including the eutrophication of rivers, the deterioration of drinking water sources, and hazards to human health. Furthermore, nitrates can form potentially carcinogenic compounds, such as nitrosamines and nitrosamides, and nitrate consumption can cause methemoglobinemia in infants. The Second National Census of Pollution Sources survey showed that the total nitrogen emission from aquaculture was 99 100 tons in 2017. To protect the environment and human health, it is important to remove nitrogen from aquaculture tailwater before discharging it to the surrounding waters. Biological denitrification is considered the most promising approach since nitrate can be reduced to harmless nitrogen gas by bacteria. A sufficient carbon source is necessary during the heterotrophic denitrification process. To solve the problems mentioned above, external liquid carbon sources such as methanol, acetic acid, and glucose are added to the tailwater, but they are costly, require high-energy, and have high operating requirement. In contrast, agricultural wastes as a carbon source have exhibited significant economic advantages and high efficiency. Many aquaculture tailwater treatment systems often face variations in hydraulic retention times (HRT) and influent nitrate concentration (INC), which are caused by acute changes in tailwater characteristics and production, and HRT and INC often exert a profound effect on the treatment performance of biological treatment systems. Extensive research has confirmed that adding agricultural waste (such as corncob, woodchip and rice straw) to municipal sewage and industrial wastewater can effectively improve denitrification efficiency. However, the effect of using agricultural waste as denitrifying carbon source to treat aquaculture tail water remains unclear. Banana stalk (BS), a typical agricultural waste product, is used as a denitrifying carbon source for the first time in this study. The study investigated the effects of HRT and INC on the denitrification performance of BS, and provided a theoretical basis for the application of agricultural waste in aquaculture tailwater treatment. In this study, using BS as a carbon source and a towel as biological carrier, the performance of solid-phase denitrification under dynamic flow conditions was studied by using a 1-D column experiment. In the HRT optimization experiment, INC was maintained at 50 mg/L and operated under four HRTs (16 h, 20 h, 24 h and 28 h) for 14 days. The effluent NO3–-N, nitrite (NO2–-N), NH4+-N, Total nitrogen (TN), Total phosphorus (TP), and chemical oxygen demand (COD) removal efficiency were measured every 2 days. The optimal HRT of BS-DR (banana stalk-denitrification reactor) was optimized by one-way ANOVA analysis. Then, based on the optimization of HRT, the reactor was operated for 14 days under different INC (75 mg/L, 100 mg/L, and 125 mg/L). The sampling time interval and measurement indexes were the same as those of the HRT optimization experiment. The Illumina MiSeq high-throughput sequencing method was used to sequence and analyze the two hyper-variable regions (V3-V4) of the 16S rRNA gene of bacteria in the initial and final stages of the BS-DR. The results indicated that HRT and INC are the key factors affecting the denitrification performance of BS-DR. There was no significant difference in nitrate removal efficiency when the HRT was 20 h (96.71±1.36)%, 24 h (94.57±4.73)%, and 28 h (99.41±0.64)%, but they were significantly higher than that when the HRT was 16 h (87.53±7.95)%. Therefore, the optimal HRT for BS-DR was 20 h, and no nitrite accumulation. The second set of experiments was conducted using the optimal HRT obtained from the first set of experiments. The effluent nitrate concentration (ENC) and nitrate removal rate (NRR) of BS-DR increased significantly with increase in INC (P<0.05), and the effluent COD decreased with increase in INC, and the proper INC for BS-DR was ≤50 mg/L. It is worth noting that BS-DR could completely remove NH4+-N in both experiments. In addition, HRT significantly affects the removal efficiency of TP, but INC has little effect. According to pyrosequencing analysis, the microbial community structure of BS-DR changed after long-term operation, with the relative abundances of Proteobacteria, Bacteroidetes, Campilobacterota, and Firmicutes increasing to 31.20%, 6.67%, 3.08%, and 4.26%, respectively, ensuring the efficient operation of the reactor. On the contrary, the relative abundances of Halobacterota, Desulfobacterota, Sva0485, Chloroflexi, and Verrucomicrobiota decreased to 10.39%, 5.13%, 2.82%, 2.00%, and 1.17 %, respectively, in the reactor. In addition, at the genus level, most of the dominant bacteria at the end of reactor operation play a role in denitrification and degradation of agricultural waste, which is significantly different from that at the beginning of the reactor operation.

Abstract:Antimicrobial peptides (AMPs) are an important part of the innate immune defense system; they can effectively kill viruses, fungi, and bacteria, preventing infection (and even sepsis). AMPs also have other functional roles in immune regulation, anti-tumor activity, angiogenesis, and wound healing. Liver-expressed antimicrobial peptide 2 (LEAP-2) is an AMP that has been extensively studied in mammals, birds, and fishes. The earliest fish LEAP-2 study reported the cloning of LEAP-2A and LEAP-2B of rainbow trout. So far, LEAP-2 has been studied in various fishes, including blunt snout bream, grass carp, golden pompano, ayu, and large yellow croaker. These studies found that LEAP-2 generally contains four highly conserved cysteine residues and two disulfide bonds; it can disrupt the structural integrity of bacterial cell membrane, revealing its antibacterial activity with an important role in the innate immune system of fishes. Russian sturgeon (Acipenser gueldenstaedtii) is cultivated on a certain scale in China, and Aeromonas hydrophila is its main pathogen. Hemorrhagic ascites and tissue hemorrhage appear after infection, causing huge economic losses to Russian sturgeon aquaculture. As an important innate immunity component, LEAP-2 research is of great significance. At present, studies on AgLEAP-2 have not been reported. To study the molecular characteristics and the transcriptional expression patterns of AgLEAP-2, as well as its antibacterial activity in vitro, the full-length AgLEAP-2 cDNA sequence was obtained by RACE. AgLEAP-2 was cloned and its sequence characteristics were analyzed by bioinformatics. The qRT-PCR method was used to detect AgLEAP-2 in 13 different tissues (liver, intestine, spleen, head kidney, blood, gill, skin, ovary, brain, heart, stomach, metanephros, and muscle) of healthy Russian sturgeon, and the AgLEAP-2 transcriptional expression pattern in immune tissues after infection with A. hydrophila. The AgLEAP-2 prokaryotic expression vector was constructed and the recombinant AgLEAP-2 protein (designated rAgLEAP-2) was purified. The antibacterial activity of rAgLEAP-2 was preliminarily detected by the agar dilution method. The results showed that the full-length cDNA of the AgLEAP-2 gene was 622 bp in length, of which the 5'-UTR was 184 bp, the 3'-UTR was 192 bp, and the ORF was 246 bp in length, encoding an 81-amino acid peptide. The molecular mass of the rAgLEAP-2 protein was predicted to be 11.2 kDa, and the theoretical isoelectric point was 9.15. AgLEAP-2 contained a signal peptide (1~25 aa) and a mature peptide (26~81 aa). The mature peptide contained four conserved cysteine residues and formed a core structure is consistent with two disulfide bonds between the Cys58-Cys69 and Cys64-Cys74. The AgLEAP-2 structure agreed with the LEAP-2 family characteristics. Moreover, the results showed that the AgLEAP-2 sequence was highly evolutionary conserved. Amino acid sequence alignment and phylogenetic analysis showed that AgLEAP-2 was clustered into a clade with the LEAP-2C of fishes, with the highest similarity with the LEAP-2C of Yangtze sturgeon and Chinese sturgeon. The qRT-PCR analysis showed that AgLEAP-2 was widely expressed in all healthy tissues, with the highest expression level in the liver, followed by the intestine and muscle, with the lowest expression in the gill. The expression patterns of LEAP-2 in different fish species were slightly different, with the highest expression level in the liver of Russian sturgeon and the intestine of large yellow croaker. The similar LEAP-2 expression patterns in different species suggested that its functions may be consistent, although it also had tissue and species specificity. The AgLEAP-2 expression reached its maximum within 72 h in the liver, spleen, head kidney, gill, and blood immune tissues. Among them, the expression AgLEAP-2 changed most significantly on the gill, which increased 5 000-fold compared with 0 h. The highest relative expression level was observed in the spleen at 72 h, corresponding to a 1500-fold induction relative to 0 h, and in the intestine at 48 h, with a 900-fold induction compared to 0 h kept constant until 72 h. In addition, the rAgLEAP-2 protein exhibited good antibacterial effects against both Gram-positive bacteria (Streptococcus sp. and S. aureus) and Gram-negative bacteria (E. coli, Vibrio anguillarum, and Shewanella spp.) in a dose-dependent manner in vitro. The rAgLEAP-2 protein at 500 μg/mL could inhibit the growth of most bacteria, significantly reducing the number of colonies on the plate. In conclusion, cloning and homology analysis revealed that AgLEAP-2 was closely related to LEAP-2C. AgLEAP-2 plays an important role in the immune response of Russian sturgeon against bacterial infection, and the rAgLEAP-2 protein could inhibit the growth of Gram-positive and Gram-negative bacteria in vitro. This study enhanced our current understanding of the nonspecific immune response in fish and provided a research basis for further studies on the antibacterial mechanism of LEAP-2 found in Russian sturgeon.

Abstract:Edwardsiella tarda is a major causative pathogen of bacterial ascites in Japanese flounder, leading to massive economic losses, and the discovery of molecular markers linked to disease resistance is an effective strategy in resistance breeding programs. The Rho GTPase family comprises small proteins with a molecular weight of 20~30 kDa. Rho GTPase family members are involved in diverse cellular processes, such as cytoskeleton, cell adhesion, vesicle transport, and proliferation. In addition, they play pivotal roles in infection by different pathogens. Rho-related GTP-binding protein Rho6 (Rnd1), a member of the Rho-GTPase family, participates in various biological functions, including neural junction formation, axonal extension, tumorigenesis, neuronal function, and apoptosis. Some members of the Rho family, such as Rac1 and Rac2, regulate immune response in grass carp, large yellow croaker, zebrafish, and half-smooth tongue sole. However, the function of Rnd1 in fish is poorly understood. Japanese flounder (Paralichthys olivaceus) is greatly affected by E. tarda infections during the breeding process. In previous studies, whole-genome sequencing and assembly of Japanese flounder were performed, and subsequently, various disease resistance genes were screened to support the improvement of Japanese flounder germplasm resources. To study the role of Pornd1 in resistance against E. tarda infection in Japanese flounder, Pornd1 was cloned and identified using PCR. The full-length Pornd1 cDNA was 699 bp, containing an open reading frame encoding a 232-amino acid protein. The predicted molecular weight of PoRnd1 was 26 kDa. Sequence and homology analyses showed that the Rnd1 protein harbors a Rho-GTP superfamily structural domain, which is highly conserved in various species. PoRnd1 shares the highest homology with Rnd1 from Hippoglossus hippoglossus (98.28%). On phylogenetic tree, PoRnd1 was clustered with Rnd1 from other fish species. The single-nucleotide polymorphism (SNP) locus associated with E. tarda resistance is located at 4 575 720 bp on chromosome 14 of Japanese flounder. The frequency of the T allele in disease-resistant families (freqT=0.92) was significantly higher than that in susceptible families (freqT=0.20). The SNP was located at the 2nd intron of Pornd1. Real-time quantitative PCR was employed to characterize the expression profiles of Pornd1 in the tissues of healthy and E. tarda-infected fish. Pornd1 expression was the highest in the heart, followed by the liver, kidney, head kidney, and spleen, but its expression was low in the skin, blood, gills, and muscle. In E. tarda-infected fish, the expression of Pornd1 mRNA decreased after 6 h, then gradually increased, and subsequently reached the highest level after 48 h in the liver, kidney, and spleen. Pornd1 expression in the kidney and spleen in the 48 h group was significantly higher than that in the 6 and 12 h groups. Furthermore, Pornd1 expression in the liver of resistant families was significantly higher than that in susceptible families. Based on its His tag, the PoRnd1 recombinant protein was purified using an Ni column and subjected to SDS-PAGE. The target band of PoRnd1 at 32 kDa was observed in the gel after Coomassie Blue staining. The PoRnd1 recombinant protein (0.5 mg/mL) was used to study antibacterial activity through the Oxford cup assay. PoRnd1 significantly inhibited the growth of Staphylococcus aureus, Escherichia coli, E. tarda, and Vibrio harveyi. In summary, Pornd1 may be closely linked to disease resistance in Japanese flounder and can thus serve as an effective gene marker for disease resistance breeding. Our findings provide a theoretical basis for further elucidating the molecular mechanisms of immunity in Japanese flounder.

Abstract:In recent years, many studies on the effects of salinity on fish growth performance have been performed. A series of research results confirmed that appropriate salinity could guarantee healthy fish growth, while under extremely high or low salinity, the fish growth would be inhibited. Therefore, salinity is one of the critical factors ensuring aquaculture success. However, most previous studies only focused on fish growth and survival, without systematically analyzing the physiological and biochemical indicators or performing regression analysis of optimal salinity, especially for the hybrid grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂). A 28-day experiment was conducted to evaluate the effects of different salinities on growth performance, body composition, digestive enzyme activities, antioxidant indices, and serum biochemical indices of the juvenile hybrid grouper [initial mean weight of (29.99±2.60) g]. This study used six salinity levels of 10, 15, 20, 25, 30, and 35, named S1 (control), S2, S3, S4, S5, and S6, respectively. During the experiment, the water temperature was kept at 25–28 ℃, DO≥6 mg/L, pH 8–9, and the contents of ammonia and nitrite nitrogen were both lower than 0.1 mg/L. Firstly, the results showed that different salinity levels had different impacts on the juvenile hybrid grouper growth performance. The weight growth rate (WGR) and special growth rate (SGR) first increased and then decreased with the rise in salinity and peaked in the S4 group, which were significantly higher than those of the S1 and S6 groups (P<0.05). The feed efficiency (FE) showed a similar tendency to WGR and SGR (P<0.05). Moreover, the survival rate had no significant difference among all groups (P>0.05). The quadratic regression model analysis based on WGR and FE indicated that the optimal salinities were 22.18 and 23.16. Secondly, the whole fish crude lipid content in the S4 group (10.86%) was significantly higher than in the S1 (9.30%), S2 (9.62%), S3 (9.79%), and S6 (9.22%) groups (P<0.05). The muscle crude lipid content in the S3 (2.84%) and S4 (2.95%) groups were significantly higher than that of the S1 (2.23%), S2 (2.34%), S5 (2.44%), and S6 (2.19%) groups (P<0.05). The whole fish crude protein content peaked in the S3 group (65.24%), which was significantly higher than that of the S5 group (63.74%) (P<0.05), with no significant differences observed among other groups (P>0.05). The muscle crude protein content and the whole fish and muscle moisture and ash had no significant differences among all groups (P>0.05). Thirdly, juvenile hybrid grouper´s intestinal lipase and trypsin activities in the S3 and S4 groups were significantly higher than in other groups (P<0.05). In addition, the intestinal amylase activity was kept at 0.19–0.21 U/(mg prot) among all groups, with no significant differences (P>0.05). Fourthly, salinity changes greatly impacted the antioxidant capacity of the juvenile hybrid grouper liver. The activities of the liver superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and catalase (CAT) were significantly higher in the S1 and S6 groups than in other groups (P<0.05). The liver malondialdehyde (MDA) content in the S3 and S4 groups were significantly lower than that of other groups (P<0.05). Fifthly, lower activity levels of the aspartate aminotransferase (AST), cereal third transaminase (ALT), and alkaline phosphatase (AKP) in serum were found in the S3 and S4 groups, which were significantly lower than those of the S1 and S6 groups (P<0.05). Moreover, the ALT activity in the S6 group was significantly lower than in the S1 group (P<0.05). The lower activity level of lactate dehydrogenase (LDH) in serum was also found in the S3 and S4 groups, which were significantly lower than in other groups (P<0.05). In summary, these results indicated that optimal salinity (20–25 mmol/L) could improve the juvenile hybrid grouper growth, while increasing the activities of digestive enzymes, antioxidant properties, and nonspecific immunity capacity, protecting the fish health. At the same time, the quadratic regression model analysis based on WGR and FE indicated that the optimal salinity for juvenile hybrid grouper is between 22.18 and 23.16.

Abstract:The acute toxicity of cadmium (Cd2+) to juvenile Thamnaconus septentrionalis was determined using a hydrostatic biological assay. According to the pre-experiment results, four Cd2+ concentration gradient test groups (8.19, 9.18, 10.30, and 11.56 mg/L) and one blank control group were selected for the acute toxicity test using the equilogarithmic spacing method. The mortality, LC50, and safe concentration of Cd2+ in juvenile fish at 24 h, 48 h, 72 h, and 96 h were calculated. Based on the experimental results of acute toxicity of Cd2+ to juvenile Thamnaconus septentrionalis, the Cd2+ concentration gradients were set as 1.84 mg/L, 2.76 mg/L, 3.68 mg/L and 4.60 mg/L at the safe concentration multiple. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione antioxidant enzyme (GSH-PX), and the content of malondialdehyde (MDA) in liver and gill tissues were observed at 6 h, 12 h, 24 h, 48 h, 72 h and 96 h. The results showed that as Cd2+ concentration increased, the acute toxicity gradually increased, the LC50 at 24 h, 48 h, 72 h and 96 h was 11.47 mg/L, 10.82 mg/L, 9.84 mg/L, and 9.19 mg/L, respectively, and the safe concentration of Cd2+ on juvenile filefish was 0.92 mg/L at 96 h. Within 6 h, SOD activity in each concentration group was significantly higher than that in the control group (P<0.01); SOD activity first decreased and then increased within 6–24 h; however, from 24 h to 96 h, SOD activity of all concentration groups showed a downward trend. SOD activity in the 1.84 mg/L and 2.76 mg/L groups was always higher than that in the control group within 96 h. The enzyme activities of the 3.68 mg/L and 4.60 mg/L groups decreased to below that of the control group from 6 h to 12 h, and increased at 24 h, after which it showed a constant downward trend and remained lower than that of the control group. From 6–12 h, MDA content first decreased and then increased. At 24 h, the enzyme content in each treatment group decreased, but was still higher than that in the control group, and the difference was significant (P<0.05). From 12 h to 48 h, the 1.84 mg/L and 2.76 mg/L groups showed a trend of first decreasing and then increasing, whereas within the 24 h to 96 h period, the MDA content in the 3.68 mg/L and 4.60 mg/L groups kept increasing, and was significantly higher than in the control group (P<0.01). At 6 h, CAT activity in all treatment groups was significantly increased when compared to that of the control group; however, CAT activity in the 3.68 mg/L and 4.60 mg/L groups was significantly inhibited at 48–96 h. Overall, the activity of GSH-PX in each treatment group showed a decreasing, increasing, and then decreasing trend within 6–96 h. The activity of GSH-PX in the 1.84 mg/L group was higher than that in the control group within 96 h. When compared with the control group, the activity of the 2.76 mg/L and 3.68 mg/L groups decreased from 24 h to 96 h, and the longer the stress time, the lower the activity. In the highest concentration group (4.60 mg/L), the enzyme activity first increased and then decreased, and the GSH-PX activity decreased to 300.12 U/mg prot at 96 h, which was significantly different from that in the control group (P<0.01). In the 1.84 mg/L group, there was no significant change in liver tissue within 72 h, but in the 1.84 mg/L group at 96 h and the 2.76 mg/L group at 24 h, the cell volume was slightly increased, cells had an irregular shape, and some cell membrane boundaries were blurred. At 48–96 h in the 2.76 mg/L group, and 24–48 h in the 3.68 mg/L and 4.60 mg/L groups, the liver cells were disordered and scattered, and the structure of liver cells was mostly incomplete. After 72–96 h in the 3.68 mg/L group and 72 h in the 4.60 mg/L group, the abnormality of liver cells was significantly aggravated, including cell hypertrophy, large area disintegration, cytoplasm overflow, and cell cavitation. At 96 h in the 4.60 mg/L group, the nucleus shrank, intracellular material gathered, and a large irregular blank appeared. For 72 h in the 1.84 mg/L group and 24 h in the 2.76 mg/L group, there was no significant change in gill tissue structure, whereas after 96 h in the 1.84 mg/L group, 48 h in the 2.76 mg/L group, and 24 h in the 4.60 mg/L group, the gill microplates were curved and club-like with mucus secreted around them, and the epithelial cells were enlarged, showed edema and partial hyperplasia, and the distance between the adjacent lamella became smaller. At 72 h in the 2.76 mg/L group, and at 24–48 h in the 3.68 mg/L and 4.60 mg/L groups, a small number of adjacent gill lamellae would fuse and form a small epithelial cell plate, with a large amount of mucus around them, and the length of gill lamellae was significantly shortened. In the 2.76 mg/L group at 96 h and the 3.68 mg/L group at 72 h, most of the basal lamellae had fused together, the epithelial cells of the gill lamellae were swollen and showed hyperplasia, and exfoliated epithelial cells were scattered around. At 96 h in the 3.68 mg/L group and 72–96 h in the 4.60 mg/L group, adjacent gill lamellae had adhered and fused with each other without a free end, and a large number of cells were necrotic and exfoliated.

Abstract:The innate immune response serves as the first line of defense and is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are ancient innate immune receptors involved in pathogen-related molecular pattern recognition, which is essential for immune homeostasis and the prevention of infection. As a member of the TLR11 family, TLR13 has been identified in several teleost fishes, including Larimichthys crocea, Oreochromis niloticus, and Epinephelus coioides. These studies have mainly focused on the function of TLR13 in protecting the body from bacterial or viral invasion. To further investigate the immune function of TLR13, the gene of open reading frame (ORF) sequence of TLR13 (ToTLR13) from golden pompano (Trachinotus ovatus) was cloned and characterized in this study. The expression pattern of ToTLR13 was determined in healthy tissues and infected immune-related tissues in golden pompano by real-time fluorescence quantitative PCR (RT-qPCR). Moreover, subcellular localization of ToTLR13 in A549 cells was determined. The results showed that the ORF sequence of ToTLR13 was 1 269 bp, encoding 422 amino acids with an isoelectric point of 8.13. ToTLR13 was classified as a hydrophilic protein by hydrophilic prediction. In addition, ToTLR13 contains a 15-amino-acid-coded signal peptide. Conservative structure domain analysis showed that ToTLR13 contains a transmembrane (TM) domain, a leucine-rich repeat (LRR) domain involved in ligand recognition and binding, and a conserved Toll/interleukin-1 receptor (TIR) domain involved in signal transduction, which is in line with the typical characteristics of the TLR family. The TIR domain exists in almost all transmembrane TLRs and its sequence is highly conserved. By establishing the tertiary structure of the conserved domain of TLR13, ToTLR13 has a high spatial structure that overlaps with the LRR and TIR domains of Mus musculus and L. crocea TLR13, which shows that the TLR13 structure and function in different species have a certain similarity. Multiple sequence alignment showed that ToTLR13 had a high similarity with other teleost fish TLR13 (82.52%~84.58%), while with other classes of species, sequence similarity was low (33.30%~46.11%). Furthermore, according to the phylogenetic tree analysis, we found that the relationship between ToTLR13 and other teleost fish TLR13 is relatively close, among which Epinephelus lanceolatus is the closest evolutionary position. While it is distant from other species, mammals are grouped into one branch; Xenopus tropicalis and Cyclina sinensis are in another branch. RT-qPCR results revealed that ToTLR13 was constitutively expressed, with the highest expression level in the gill and spleen, followed by the brain, liver, and kidney, and expression was lower in the heart, head kidney, and muscle. The mRNA expression of TLR13 is slightly different in different fish, which indicates that TLR13 has species specificity and tissue specificity in normal fish tissues. Moreover, TLR13 is generally highly expressed in fish immune-related tissues, suggesting that TLR13 may play different roles in different fish species and plays an important immunomodulatory role. When stimulated by pathogens or viruses, the mRNA expression of TLR13 in immune-related tissues of different fish varies. In this study, after infection with Streptococcus agalactiae and Vibrio alginolyticus, there were significant changes in the mRNA expression of ToTLR13 in different tissues. The ToTLR13 mRNA expression level in the gill suddenly reached a peak at 72 h after infection with S. agalactiae, but showed significant differences at 12 h and 96 h in the V. alginolyticus experimental group. In the spleen, the mRNA expression of ToTLR13 increased in a time-dependent manner after infection with S. agalactiae and V. alginolyticus, peaking at 24 h and 96 h, respectively. The mRNA expression level of ToTLR13 in the liver showed a regular trend of increasing and then decreasing from 0 h to 48 h after S. agalactiae infection and reached a peak at 72 h. In the V. alginolyticus experimental group, the mRNA expression level of ToTLR13 in the liver decreased to below the original level at first and then increased, reaching a peak at 48 h. In the kidney, the ToTLR13 mRNA expression level in the S. agalactiae group reached a double peak at 12 h and 72 h after infection, respectively. ToTLR13 mRNA expression level reached a peak at 6 h after V. alginolyticus infection, and then decreased to the level before challenge. These results suggest that ToTLR13 plays an important role in the immune response against pathogenic bacteria. According to their intracellular localization, TLRs can be divided into two categories: Those expressed on the surface of the cell membrane and those localized in the cytoplasm. In this study, subcellular localization showed that ToTLR13 was localized in the cytoplasm of A549 cells, and this phenomenon was also found in other teleost fish TLR13. The results of this study showed that ToTLR13 might be involved in the innate immunity of golden pompano against pathogenic bacteria, which lays a foundation for studies on the function of TLR13 in teleost.

Abstract:Fish are the main source of long-chain polyunsaturated fatty acids (LC-PUFA), in particular docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), which are nutritionally valuable to humans. Moreover, lipid and fatty acid distribution in fish tissues are highly diverse across species. The lipid distribution pattern across tissues determines fatty acid profile, one of the product quality indicators of fish. However, this diversity of lipid distribution and fatty acid profile is not fully known, although several studies have been conducted to compare the nutritive composition of muscle in different fish species in both freshwater and marine environments. This study aimed to comprehensively investigate the lipid distribution and fatty acid profile, as well as the somatic indexes and approximate composition of five benthic marine fish species, namely yellow anglerfish (Lophius litulon), flathead (Platycephalus indicus), eelpout (Zoarces viviparus), East Asian flatfish (Pleuronichthys cornutus), and zebra sole (Zebrias zebrinus). Fishes were purchased from the 15th Street Seafood Market of Qingdao, which were stored on ice since captured in an inshore fishery in Qingdao (located at 119°30'–121°00'E and 35°35'–37°09'N), and then delivered to the market from the fishing harbor in the same morning. After purchase, fishes were kept on ice and immediately taken to the laboratory. The experimental design consisted of nine fish of each species bulked in groups of three, comprising three replicates. First, the total weight and length of each fish were measured, and the liver and viscera weight were recorded to calculate the viscerosomatic index (VSI), hepatosomatic index (HSI), and condition factor (CF). Subsequently, samples of muscle, liver, brain, eye, skin, subcutaneous adipose tissue around fins (only for East Asian flatfish and zebra sole), and intraperitoneal adipose tissue (only for flathead) were collected for lipid and fatty acid analysis. The dorsal muscle sampling position is behind the head on the right body side. The ventral muscle samples were collected under the dorsal muscle sampling spot. The proximate composition analysis of the dorsal muscle, ventral muscle, and liver (three individual samples per group) was performed according to the Association of Official Analytical Chemists standard methods. For the moisture assay, samples were oven-dried at 105 ℃ until they achieved a constant weight. The protein content was assayed by measuring nitrogen (N × 6.25) using the Soxhlet method, the lipids were analyzed using the Soxhlet method (petroleum ether extraction), and the ash by incineration at 550 ℃. The fatty acid compositions of all tissues were analyzed with gas chromatography (GC-2010, Shimadzu, Japan). The results showed that the flat body type of flatfish determines that the gut in these fish species cannot store high lipid contents in the liver or intraperitoneal adipose tissues. Like other flatfish, East Asian flatfish and zebra sole had lower HSI and VSI. In contrast, the HSI and VSI of yellow anglerfish and flathead were associated with high lipid contents in the gut. Additionally, the lipid distribution across tissues indicated that yellow anglerfish and flathead predominantly stored lipid in the liver and intraperitoneal adipose tissue, respectively. The muscle lipid content of all the five species was low, especially for yellow anglerfish and zebra sole (0.3%–0.4%). Yellow anglerfish, flathead, and East Asian flatfish had higher liver lipid content (19%–29%) than zebra sole and eelpout (4%–5%). Yellow anglerfish and flathead store lipid predominantly in the liver and intraperitoneal adipose tissue, respectively. The n-3 LC-PUFA content, especially DHA, in the muscle of yellow anglerfish was significantly higher than that of other species. The yellow anglerfish dorsal muscle also had the highest 18:1n-9 content. The relative abundances of different monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) in different fish species indicate a preference for energy storage and lipid mobilization. The most distinct fatty acid characteristics of the dorsal muscle of eelpout were low 16:0 content and high EPA content. For liver fatty acids, flathead and East Asian flatfish had significantly high MUFA contents, such as 18:1n-9, but their DHA contents were low. High DHA, 20:4n-6, and 22:5n-3 contents were consistently observed in nearly all yellow anglerfish, eelpout, and East Asian flatfish tissues, respectively, while low EPA content was observed for all of them. Among all five fish species, eelpout had a much higher ARA content than the other species in nearly all tissues. This could be due to the high ARA level in the food sources, namely algae. Yellow anglerfish had significantly high n-3 LC-PUFA content and thus high fatty acids nutritional value. This study revealed that even though these fishes are all benthic species, there are great differences in lipid and fatty acid composition among them.

Abstract:As low-level aquatic vertebrates, fish are highly dependent on the water environment, and general activities such as growth, foraging, and reproduction are easily affected by changes in the external environment. Changes in environmental factors can lead to different degrees of stress response in fish, and trigger a series of physiological changes, which then affect the stability of the organism's internal environment. Predation is one of the main environmental factors affecting the survival of individuals. In nature, almost all species face the risk of predation. Brief encounters with predators can reduce feeding and other health-related activities in prey fish and/or trigger primary and secondary stress responses, including the release of stress substances into the bloodstream. In predation stress, after initially sensing stress, fish initiate a stress response to overcome the stress and restore homeostasis. The degree of physiological stress depends primarily on the intensity and duration of the stress. If the appearance of predators is intermittent, then the physiological state of the prey fish returns to normal quickly, which allows the stress response to promote physiological changes in the prey fish to better adapt to the environment. However, repeated or persistent and unavoidable stress situations cause the normal physiological response mechanisms of prey fish to become compromised. Physiological stress may have long-term negative effects on the immune system, growth, or reproduction, and may reduce the adaptability and survivability of prey fish in the environment. Many studies have confirmed that predation stress can cause physiological stress in fish. Different species of fish and even different groups of the same species vary greatly in the degree of stress and stress mode. More species-specific studies are required to determine the effects of different levels of predation stress on physiological stress in fish. Black carp (Mylopharyngodon piceus), grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys molitrix), and bighead carp (Aristichthys nobilis) are known as the four major Chinese carps. As common fish species in Chinese inland watersheds, the four major Chinese carps are ecologically and economically valuable. Over the years, many reasons such as hydraulic construction, environmental pollution, and overfishing have led to sharp declines in wild populations. In addition, the prevalence of predators in natural waters also threaten population growth. It remains unclear how the juveniles of the four major Chinese carps adjust their physiological processes to cope with predation stress. We investigate the physiological and energy metabolism adaptations by black carp, grass carp, silver carp, and bighead carp to predatory stress. We selected the common local enemies of natural waters, the snakehead carp (Channa argus) and the southern catfish (Silurus meridionalis) as predators. The levels of serum cortisol and biochemical parameters in the juveniles of the four major Chinese carps under the stress of no-predation (control), low-predation (indirect stress) and high-predation (direct stress) over 0 d, 7 d, and 14 d were investigated. Changes in the biochemical parameters were analyzed. The effects of different predation stress levels on serum cortisol and biochemical parameters were also analyzed. The results showed: (1) under different levels of predation stress, the biochemical parameters and serum cortisol levels of juveniles of the four major Chinese carps varied to different degrees but the trends were consistent; (2) the serum cortisol levels of juveniles of the four major Chinese carps increased significantly with the degree of predation stress and the stress duration, and showed the following patterns: non-predation group < low predation group < high predation group, 0 d < 7 d < 14 d. (3) Among the biochemical parameters, serum total protein concentration and cholesterol concentration were relatively stable and did not vary significantly. Glucose concentration and alkaline phosphatase increased with predation stress, while triglyceride had a decreasing trend. The results showed that juveniles of the four major Chinese carps adjust their physiological responses to enhance their own survivability according to the predation risk. After the predation stress treatment, the juveniles of the four major Chinese carps all underwent a stress response. Compared with indirect predation, direct predation had a more significant effect on the physiological response of fish, and the degree of stress increased with the stress duration. Among the detection parameters, serum total protein and cholesterol may not be sensitive parameters for stress in fish under predation stress. The most significant changes were in cortisol and glucose and may compensate for the increased energy demand by the organism during stress. The adaptation of physiological stress and energy metabolism to predation stress in juveniles of the four major Chinese carps under predation stress conditions provides a theoretical basis for the stress responses of an organism to environmental changes, and can also provide a scientific reference for exploring the ecological interactions between predator and prey.

Abstract:Diel variation of dissolved oxygen content in aquaculture is common. Generally, the dissolved oxygen content in the pond is the highest in the afternoon and the lowest in the early morning. Siniperca chuatsi usually feeds in the early morning because of its unique feeding habits, therefore the low dissolved oxygen level may affect its metabolism. In this study, S. chuatsi was subjected to different oxygen conditions, a control group (>5 mg/L) and an experimental group (dissolved oxygen changed with sunlight) for 24 h to determine glucose metabolism systemically. The following results were obtained: (1) Dissolved oxygen level in the experimental group that underwent periodic variation was the lowest at 07:00, with an average dissolved oxygen level of 0.93 mg/L; and the dissolved oxygen was the highest at 17:00, with an average dissolved oxygen level of 10.58 mg/L. (2) At T3 and T4, the plasma content of experimental group significantly decreased and lactic acid content increased significantly; liver glycogen and ATP content decreased, whereas lactic acid content increased significantly. (3) At T3 and T4, the activities of the key enzymes involved in glucose metabolism in liver varied; the activity of aerobic metabolism enzyme citrate synthase (CS) significantly decreased while that of anaerobic metabolism enzyme lactate dehydrogenase (LDH) increased significantly; the activity of glycolysis enzyme hexokinase (HK), phosphate fructose kinase (PFK), pyruvate kinase (PK) increased significantly and the activity of glycogenolytic enzyme glycogen phosphorylase (GP) exhibited no significant difference. (4) At T3 and T4, the expression of hk, pfk, pk, ldh-a, gp genes in the experimental group increased significantly, the expression of cs significantly decreased, and the expression of glucose transporter 2 (glut2) was not significant difference. Thus, when diel variation periodically changed dissolved oxygen level in the pond, lower dissolved oxygen level lead to change in glucose metabolism, from aerobic metabolism to anaerobic metabolism, and the energy utilization mode of S. chuatsi had changrd.

Abstract:Litopenaeus vannamei is an important aquaculture species in China. Selective breeding is an effective means to improve the economic benefits of aquatic animals. In terms of the energy budget, water temperature is the key factor affecting the metabolism of aquatic invertebrates at different stages. It affects their survival and overall growth and may become a fatal environmental driver. Therefore, in recent years, the impact of temperature on the energy budget of important economic aquatic animals has attracted extensive attention. Taking L. vannamei as an example, the energy budget under constant and variable temperature conditions was studied. In order to further develop the breeding of L. vannamei and clarify its energy distribution mode under different temperature conditions, this study used three families of L. vannamei (N310010, N310004, and N310011) as materials and measured their individual energy budget at the temperature of 20 ℃ to 35 ℃ during 40 days. The physiological indexes such as feeding, oxygen consumption, ammonia excretion, and fecal excretion rates were measured every ten days. The individual energy budget of L. vannamei families with different specifications (S1, S2, S3, and S4) was analyzed. The results based on the range temperature of 20 ℃ to 35 ℃ showed the total specific growth rate of 9.79% for the N31004 family, which was significantly higher than that of the other two families (P<0.05). In the S1~S2 period, the specific growth rate of the N31004 family was significantly higher than that of other families (P<0.05), in which the highest rate was 27.76%. In the S2~S3 period, there was no significant difference between the N31004 and N310011 families, while the N31004 family growth rate of 5.79% was significantly higher than that of the other two families, while the lowest was 3.07% observed in the N310011 family. Moreover, the highest feeding rate [0.026 g/(g·d)] was found in S1, and the lowest [0.005 g/(g·d)] was observed in S4. Overall, the feeding rate of L. vannamei with different specifications and families was significantly different (P<0.05). The feeding rate of the N31004 family was higher than that of the other two families at 20 ℃ in the S1 period, whereas the feeding rate of N310004 family was significantly higher than that of the other two families in the S2, S3, and S4 periods. Furthermore, in N310011, the oxygen consumption rate was significantly higher than in the other two families at 25 ℃ to 35 ℃ in the S1 period and 35 ℃ in the S2 period. There was no significant difference between the oxygen consumption rate of the other two families (N310010 and N31004) under these conditions. The N310010 oxygen consumption rate was significantly higher than the other two families at 30 ℃ to 35 ℃ in the S3 period and 20 ℃ to 35 ℃ in the S4 period. In the S1 to S3 periods, the N31004 oxygen consumption rate was significantly lower than the other two families at all temperatures. In the S4 period, there was no significant difference among the three families at 30 ℃ to 35 ℃. Overall, the ammonia excretion rate of the N31004 family was significantly lower than the other two families. Finally, the maximum feeding energy of the N31004 family was 1 510.62 J/(g·d) at 35 ℃ and the minimum was 812.47 J/(g·d) at 20 ℃, which were significantly higher than those of the other two families at all temperatures. N310010 showed the highest respiratory energy at 35 ℃ [1 061.15 J/(g·d)] and the lowest at 20 ℃ [566.18 J/(g·d)], which was significantly higher than the other two families. The excretion energy of N310010 family was significantly higher than that of the other two families at each temperature. The growth energy of the N31004 family was the highest at 30 ℃ [298.45 J/(g·d)] and the lowest at 20 ℃ [163.61 J/(g·d)]. The defecation energy of the N310010 family was significantly lower than that of the other two families. Compared with the other two families, N31004 had more energy for growth, although the respiratory excretion energy was relatively low. It is of great significance for the L. vannamei breeding to deeply understand the effects of physical and chemical environmental factors such as temperature on the growth, development, and energy budget of different L. vannamei families from a physiological and ecological perspective, clarifying the L. vannamei energy distribution mode under different temperature conditions. Currently, there are no reports on the energy basis and temperature effect of the growth rate of diverse L. vannamei families. Therefore, by establishing energy distribution models, this study explores the impact of temperature on the energy budget and distribution of L. vannamei and provides theoretical and experimental support for healthy culture and breeding of improved L. vannamei varieties.

Abstract:Litopenaeus vannamei is one of the important crustaceans in aquaculture in China. In recent years, the shrimp culture industry has gradually developed into an intensive and high-density model, which can lead to environmental deterioration and large-scale outbreak of diseases. The widespread use of antibiotics has led to the increase of pathogen drug resistance, environmental pollution, and ecological imbalance, resulting in secondary pollution in the water. Probiotics are live microbial additives that promote good health and are environmentally sustainable and safe for use. They are often used as important substitutes for antibiotics. Brevibacillus laterosporu, as a biocontrol probiotic, is commonly used for disease control of crops, animals, and poultry, but its use is rarely reported in aquaculture. A 28-day breeding experiment was carried out with L. vannamei weighing (1.00±0.08) g, to study the effects of adding B. laterosporu FAS05 in the feed on the growth, disease resistance, and immunity of L. vannamei. The experiment was divided into four groups with three replicates in each group, and 50 shrimps in each replicate were fed with the experimental feed supplemented with 0 CFU/g (group C, as the control group), 105 CFU/g (group BL1), 107 CFU/g (group BL2), and 109 CFU/g (group BL3) of B. laterosporu FAS05. B. laterosporu FAS05 was isolated from the aquaculture pond with a low number of Vibrio in summer. The bacterium was added into the basic feed with fish meal, soybean meal, and corn meal as the main protein source, fish oil and phospholipid oil were added as the fat source, wheat flour as the main sugar source, and inorganic salts and vitamins as supplements. L. vannamei was purchased from a prawn farm in Weihai City, Shandong Province. The temperature was 25–28 ℃ and the salinity was 27–30. During the breeding experiment, eight shrimps were randomly selected in each tank every two weeks and their body length, weight, and plumpness were measured. The phagocytic activity assay was modified on the basis of the method reported by Delaporte et al (2003). Fl-1 channel flow cytometry was used to detect and analyze the offset of respiratory burst peak. The blood lymphocytes, serum, and hepatopancreas of shrimp were collected 24 hours after the experiment. The activities of immune related enzymes in hepatopancreas, such as superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), and alkaline phosphatase (ALP) levels, and the activities of immune related enzymes in serum, phenol oxidase (PO), and lysozyme (LZM), were determined by kits. The phagocytic activity was measured, and the assay was modified on the basis of Delaporte et al (2003). Fl-1 channel flow cytometry was used to detect and analyze the offset of respiratory burst peak. After the feeding test, a one-week infection test of Vibrio parahaemolyticus was carried out to determine disease resistance. The experimental results were expressed as Mean±SD. One-way ANOVA analysis was conducted for all data using statistical software SPSS 16.0, and significance level was defined as P<0.05. LSD homogeneity of variance test was used to compare the differences between experimental treatment groups and control groups. The results showed that there was no significant difference in survival rate among all groups (P>0.05), and the body length, body weight, and specific growth rate of shrimps in the BL1 and BL2 groups were significantly higher than those in group C (P<0.05). Compared to that in group C, the abundance of Vibrio in the aquaculture water in the BL1 to BL3 groups was significantly decreased (P<0.05). After infection with V. parahemolyticus, the survival rate of group C was about 45%, while the survival rate of the BL1 to BL3 groups was more than 80%; therefore, the survival rate of the BL1 to BL3 groups was significantly higher than that in group C (P<0.05), and no significant difference was found among the BL1 to BL3 groups (P>0.05). Compared to that of group C, the phagocytizing rate of shrimp blood cells in the BL1 to BL3 groups increased significantly (P<0.05), while the production of ROS in blood cells decreased significantly (P<0.05). The LZM, CAT and ACP activities of shrimps in the BL1 to BL3 groups were significantly higher than those in group C (P<0.05). There was no significant difference in LZM and ACP activities among the BL1 to BL3 groups (P>0.05). The PO of shrimps in the BL2 group was significantly higher than those in other groups (P<0.05). The ALP and SOD in the BL1 and BL2 groups were significantly higher than those in group C and BL3 (P<0.05). The above results showed that B. laterosporu FAS05 as a feed additive could promote the growth of shrimp, activate the immune system, improve disease resistance, and inhibit the growth of Vibrio in the surrounding environment. The reference dosage was 105 CFU/g. When the infection is serious, the dosage can be increased to 107 CFU/g to further improve the non-specific immunity of shrimp. The results of this study can provide basic data for the application of B. laterosporu FAS05 in shrimp culture and production.

Abstract:Mytilus galloprovincialis has high-protein and low-fat contents, as well as medicinal and therapeutical values. It is one of China's most important mussel breeding species, with strong adaptability, mature and straightforward breeding technologies, and high yield. It mostly feeds on small plankton, with main nutrients varying seasonally with different growth stages, and the individual biochemical components of the same species also differ geographically. The change of bait is also an important factor in changing the volatile substances of this shellfish. The mussels' nutritional flavor characteristics have been a concern of national scholars. However, the nutritional and flavor characteristics of M. galloprovincialis in different cultural areas have not been reported yet. Haizhou Bay is the main M. galloprovincialis aquaculture area in China, with an annual output of more than 150 000 tons and a value of 300 million yuan. Mussels cultivated in five sites (H1 to H5) in this region were collected once every month, from January to March. H1 is one nautical mile off the coastline; H2 is 3, H3 is 7, H4 is 13, and H5 is 21 nautical miles off the coastline. The physical and chemical factors of these water environments and the protein, fat, total sugar, and glycogen contents within the soft tissues of the sampled mussels were measured. The results showed that the M. galloprovincialis nutrient content varied among sampling time and sites and was directly related to the gonad development. The H1 condition index increased continuously from January to March, and the mussels' highest condition index in other areas was found in February. The highest total sugar content of mussels collected in all regions was observed in February, while for the H4 and H5 areas, it decreased significantly in March and was 62.7% and 61.6% lower than that in February, respectively. These changes were mainly caused by M. galloprovincialis entering its breeding season in March. The total content of 16 amino acids and the content of flavorful amino acid at H5 were significantly higher than those in other regions in January (P<0.05). However, these amino acid levels decreased significantly and became the lowest in March for the mussels sampled in the H5 region. From January to March, the glycogen content in the mussel mantle increased firstly and then decreased, while its highest level was found in mussels collected in H5 in March. Most of the cultivated M. galloprovincialis are sold in winter. Here we observed that the gonad development and nutrients and flavor amino acids levels of the offshore mussels were different from those of the nearshore mussels, and the offshore mussel completed the material and energy storage earlier and entered the breeding period sooner, suggesting that the best marketing time is different for the studied areas. Differences in the mussels´ nutrient reserve were mainly related to temperature and feed density. The water temperature and the Chlorophyll-a (Chl-a) concentration tended to increase with the offshore distance rise, leading to the rapid development and early marketing time of M. galloprovincialis in the studied offshores. In February, mussels in the H5 area were rich in nutrients and flavor materials; therewith, they could be marketed one month earlier than in other areas. Early listing of the offshore cultured mussel can prolong its market supply time and reduce the competitive pressure caused by centralized listing. In addition, the offshore cultured mussel has a good market potential because of its short breeding cycle, wide breeding space, and good taste. In recent years, thanks to the development of internet technology, online sales have become an important sales channel in all industries. This way avoid the third-party vendor, wherein the producer can sell directly to customers with higher unit prices and a vast market. Combined with the advantages of offshore mussel cultivation, farmers can use online sales to increase their income further. With the increase in the density of offshore aquaculture in the Haizhou Bay, harmful algal blooms threaten the safety of shellfish products. Environmental problems in nearshore areas are the main factors hindering the favorable and productive development of the M. galloprovincialis industry. Therefore, the development of offshore aquaculture has great potential. Compared with the nearshore sea, the offshore sea has more extensive space, frequent water exchange, and larger environmental capacity. In order to improve the output of mussel aquaculture, New England began to transfer the aquaculture area from the nearshore to the offshore sea. Although the cost and technology needed for the offshore culture are significant, the comprehensive benefits of the offshore culture are still considerable. The offshore aquaculture development is conducive to optimizing the spatial structure of the marine industry, improving the overall efficiency of food production, alleviating its resource and environmental pressures, and cultivating new marine products with technological and industrial advantages. This study may provide supportive data for optimizing the spatial layout planning and harvesting time of M. galloprovincialis offshore cultivation.

Abstract:In recent years, continuous high temperature and marine heat waves in summer have become major limitations for the sustainable development of sea cucumber aquaculture. In 2019, our team invented a cooling equipment based on cooled atomization air, which can reduce the water temperature of the culture pond in summer. Previous tests showed that the survival rates of the sea cucumbers in the equipped ponds were higher than the unequipped ponds during the high temperature. In this study, five equipped ponds were selected as experimental groups (groups E1 and E2 were labeled according to their geographic location) and three unequipped ponds were selected as control groups (labeled group C), then the water quality, sedimental quality, and the sedimental microbial community structure of the eight ponds were analyzed in order to reveal the mechanism of the higher survival rate using the cooling equipment. The results showed that the temperature of the bottom water of the two experimental groups was significantly lower than that of the control group (P<0.05). The dissolved oxygen (DO) level in the bottom water of the experimental groups was significantly higher than that of the control group (P<0.05). Both the ammonia nitrogen concentration in the bottom water and the concentrations of nitrite nitrogen, and chemical oxygen demand (COD) and ammonia nitrogen in pond sediments of the two experimental groups were significantly lower than that of the control group (P<0.05). The sedimental microbial community structure of all the ponds was also investigated, and the OTUs (optical transform units) were numbered from 707 to 808. Alpha diversity analysis showed that the abundance and diversity of the experimental group were higher than those of the control group. PCoA (principal coordinates analysis) showed that the bacterial composition and community structure among the two experimental groups were more similar, while there were significant differences between the experimental and the control groups. The species distribution analysis on the order level showed that the similarity between the two experimental groups is higher than that between the experimental and control groups. The relative abundance of Rhizobiales, Lactobacillales, and Micrococcineae in the experimental groups were significantly higher than that in the control groups. Thirteen OTUs with significant differences among three groups were selected using LEfSe (LDA effect size) analysis. The correlation analysis between microbial community structure and environmental factors showed that the abundance of microbial species involved in the nitrogen cycle was significantly higher in the experimental groups than that of the control groups. Then OTU7, OTU29 and OTU108 were screened and significant correlation was found with ammonia nitrogen concentration in all of the tested ponds, and they are classified as Ochrobactrum, Escherichia-Shigella and Bacillus, respectively. In the 25 predicted COG (clusters of orthologous groups of proteins) metabolic pathways in prokaryotes, 18 metabolic pathways exhibited significant differences (P <0.05) between the experimental groups and the control groups. All the results indicated that the use of the equipment could significantly improve the pond water quality and sediment environment, and positively affect the sedimental microbial community structure of the bottom water. The results of this study would provide scientific support for the popularization and application of the equipment.

Abstract:As a raw material for aquatic feed, kelp has a large output and is rich in sugars, minerals, vitamins, free amino acids, fatty acids, etc. It is not only a natural bait for sea cucumbers in natural seas but also often used as a main ingredient in compound feed for sea cucumbers. However, kelp contains non-starch polysaccharides (NSP) which are difficult to be digested by endogenous enzymes secreted by aquatic animals. Enzymatically hydrolyzed kelp (EKP) powder is made by hydrolyzing kelp powder with a compound enzyme preparation (NSP enzyme∶cellulase∶neutral protease∶flavor enzyme = 8∶12∶3∶1), and the compound enzyme is added at 3% (by weight). The ratio of material to liquid is 1∶6, and the conditions of enzymatic hydrolysis comprise a temperature of 50 ℃, pH 6, and reaction time 6 h. A feeding trial was conducted to investigate the effects of EKP on the growth, digestion, metabolism, and oxidation resistance of juvenile sea cucumbers Apostichopus japonicus (Selenka, 1867). In this experiment, fish meal, kelp meal and EKP were used as the main protein sources to prepare a basic feed with a crude protein content of 12.00%, a crude fat content of 0.40%, and an energy content of 6.20 KJ/g. A total of 540 healthy sea cucumbers with an initial average weight of (11.4±0.04) g were selected and randomly assigned to 18 cylindrical circulating buckets. They were divided into 6 experimental groups with 3 replicates in each group and 30 sea cucumbers in each replicate. Six experimental diets were formulated with the graded levels of EKP, 0% (D1, control group), 3% (D2), 6% (D3), 9% (D4), 12% (D5), and 15% (D6) dry diets. The feeding period was 56 days. Bait was fed once a day at a fixed time (16:00). The water was changed every 3 days, and a siphon was used to withdraw the residual bait and feces from the bottom of the bucket. The amount of water changed was 1/2 of the water level in the bucket. The breeding experiment was run for 1 month. During the breeding period, the water temperature was 13~17 ℃, pH was 7.5~8.2, dissolved oxygen was at least 6 mg/L, and salinity was maintained at 28~30. The results showed that: (1) The weight gain rate and specific growth rate of sea cucumbers in the D3, D4, D5 and D6 groups were significantly increased as compared to that in the control group, with the D4 group reporting the highest values. (2) The contents of water, crude protein, crude lipid, and ash in the body wall of sea cucumbers were not different among groups, but addition of EKP significantly increased the contents of methionine and cysteine. (3) The activities of intestinal amylase and protease increased first and then decreased with the EKP addition level increasing, but the lipase activity was not significantly affected. (4) Dietary incorporation of EKP significantly increased the apparent digestibility of crude protein, gross energy, total phosphorus and six amino acids. (5) The activities of intestinal glucokinase, pyruvate kinase, and phosphoenolpyruvate carboxykinase as well as the total antioxidant capacity increased first and then decreased; the activities of Na+-K+-ATPase or Ca2+-Mg2+-ATPase showed an increasing trend with increasing EKP addition levels. The malondialdehyde content followed a trend opposite to the total antioxidant capacity. In conclusion, appropriate addition of EKP has positive effects on growth, digestion, metabolism, and oxidation resistance of A. japonicas, and it promote the growth of sea cucumbers. The predicted optimal supplemental level of EKP in the diet of A. japonicus was 10.36% (12% feed crude protein) according to the quadratic regression analysis on weight gain rate against the EKP addition levels. This research provides a theoretical basis for the use of EKP at compound feed for A. japonicus.

Abstract:Marine fish are rich in minerals (calcium and iron), vitamins (riboflavin and niacin), nutrients that are beneficial for brain (fatty acids), docosahexaenoic acid that prevent cardiovascular and cerebrovascular diseases, and are an important source of protein for humans. In recent years, the nexus between the increased demand of high-quality protein and the decrease in marine fishery resources resulted in the intensification of marine farming. However, this high-density farming has led to various bacterial diseases frequently due to lack of disease control methods, which has caused economic losses in the farming industry and impeded the healthy development of the industry. Therefore, it is particularly important to reduce the rates of bacterial diseases and improve the survival rate of cultured fish. It is of particular concern on how to maintain intestinal health of cultured fish under this farming method. Probiotics, a kind of living microorganisms, are beneficial to the health of the host. In the 1980s, probiotics have been viewed as an environmentally friendly and effective product in aquaculture; they improve the host health by influencing intestinal microbiota and nonspecific immunity to increase disease resistance ability. Moreover, they can be used as an ideal substitute for antibiotics in aquaculture production. Probiotics from Bacillus, lactic acid bacteria, saccharomyces, and nitrobacteria are diverse, and the function of each varies widely. As the typical representative of probiotics, lactic acid bacteria, may inhibit the growth of pathogens by the production of its metabolites, such as lactic acid, acetic acid, peroxide hydrogen, and bacteriocin. Lactic acid bacteria are also able to balance intestinal microecological imbalance and maintain intestinal microbiota balance when the intestinal microbiota contains pathogenic bacteria or the host is treated with antibiotics. Bacillus is an aerobic or facultative anaerobic gram-positive bacteria, which is stable, possesses strong stress resistance and high resurrection rate, and can produce various macromolecules, such as proteases and amylases and thus can improve the digestive function of the host by promoting the absorption of nutrients. Furthermore, as a non-specific immune antigen, Bacillus can improve the immune resistance of the host by stimulation of the components of cells or cell walls. The application of lactic acid bacteria and Bacillus has demonstrated favorable results, but this has been limited in marine fish culture because the non-fish origin of some strains and the different specificity of strains for different fish species or the same growth stage make their application difficult. Thus, it is essential to develop marine fish-derived probiotics, analyze their characteristics, define growth conditions, verify safety effects, and determine dosage and methods for their administer in marine fish culture. In this study, probiotics from marine fish were screened for the development of microbial ecological agents; 80 strains of culturable bacteria were obtained by separating bacteria from the mucosa samples of the digestive tract of wild Sebastes schlegelii and Hexagrammos otakii. The enzyme-producing ability of the strains was determined using a selective culture medium. The common pathogenic bacteria infecting marine fish were selected as indicator bacteria to determine the antibacterial activity of the isolated strains. Two potential probiotics, strains TS2 and TH8, were screened, and their physiological and biochemical identification, 16S rDNA sequence, growth characteristics, and host safety were determined. The results showed that TS2 had the strongest ability to hydrolyze starch, protein, and fat, and its sterile culture products could significantly inhibit the growth of Vibrio anguillarum, V. parahaemolyticus, V. Harvey, and Pseudoalteromonas nigrifaciens. TH8 has the strongest ability to hydrolyze protein and fat, and its sterile culture products could significantly inhibit the growth of V. anguillarum, V. alginolyticus, V. parahaemolyticus, P. nigrifaciens, Aeromonas hydrophila, Staphyloccocus aureus, and Escherichia coli. According to the analysis of the physiological and biochemical characteristics of the bacteria and 16S rDNA sequence alignment analysis, strain TS2 was identified as Bacillus subtilis and strain TH8 as Vagococcus fluvialis. Strain TS2 showed significant growth at 15–40 ℃, sodium chloride concentration of 0–0.08 g/L, and pH of 5–9; it entered the logarithmic phase after 6 h and the stable phase after 26 h. TH8 grew rapidly at 20–40 ℃, sodium chloride concentration of 0–0.08 g/L, and pH of 5–12; it entered the logarithmic phase after 2 h and the stable phase after 14 h. The safety of strains TS2 and TH8 was analyzed on a homologous host, and it was found that the strains were relatively safe for the homologous host at the concentration of 108 CFU/mL. The screened B. subtilis TS2 and V. fluvialis TH8 strains have a strong enzyme production ability and inhibit the growth of various pathogens by their metabolites; they have the following advantages: Wide temperature and salt tolerance, acid and alkali resistance, and fast growth speed. Thus, they could be considered as potential probiotic candidates for the development of microbial pharmaceuticals and can be used in more applications in marine aquaculture.

Abstract:To study the regulatory role of marine bacterial motility on biofilm formation and settlement of marine bivalves, this study used the economically important marine mussel Mytilus coruscus as the research target, and investigated the wild-type strain and ΔcheW strain of Pseudoalteromonas marina. These bacteria have different motility; thus, the motility analysis of these strains was carried out at various time points, and the differences in the biofilm thickness and composition and bacterial density were also analyzed at different time points. Finally, their effects on dynamic biofilm succession and settlement of M. coruscus were evaluated. This study found that the motility of the wild-type strain and the ΔcheW strain at 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h was significantly different (P<0.05). It was found that both bacterial circle radii increased with time and reached their maximum at 96 h. Overall, the wild-type strain formed a larger bacterial circle than the ΔcheW strain. The two strains' bacterial density and biofilm thickness increased with time and reached their maximum at 48 h, begging to spread after 72 h. At all time points, the mussel settlement rates on the wild-type strain biofilm were higher than on the ΔcheW biofilm. Initially, the mussel settlement rates increased, but after 72 h a decreasing tendency was observed, reaching the highest value at 48 h. These settlement results were consistent with the changes in the biofilm composition under different time points. Therefore, this study concluded that bacterial motility affects biofilm formation, mainly biofilms thickness, bacterial density, and extracellular compounds during the dynamic succession of biofilms, thereby regulating the settlement of the mussel M. coruscus. This study provides novel insights into the interaction between bacterial motility, biofilm formation, and the settlement of M. coruscus, which may help optimize the production and breeding of economically important marine animals.

Abstract:The photovoltaic fishery breeding is a new environmentally friendly breeding method that combines pond aquaculture and photovoltaic power generation. Combining Chinese mitten crab (Eriocheir sinensis) farming with photovoltaic power generation is a new attempt to utilize resources comprehensively. However, there is no research on the effect of photovoltaic fishery breeding on the odor formation of E. sinensis. In this study, we compared the differences in basic nutrition composition, sensory evaluation, and odor quality of E. sinensis cultured in photovoltaic fishery breeding (PM) and common pond breeding (CM) environments. Two kinds of female crabs under PM or CM had their hepatopancreas and gonads evaluated by sensory analysis, as well as their tissue indices determination, proximate composition, electronic nose, and content of volatile compounds were evaluated. The flavor-active substances (odor activity value, OAV > 0.1) generated by the two breeding methods were evaluated. The results of artificial sensory evaluation and volatile compounds determination were analyzed by partial least squares (PLS) to comprehensively evaluate the odor characteristics of the hepatopancreas and gonads of Chinese mitten crab. The results of tissue indices and proximate composition showed that the gonads index of female crab was increased by the photovoltaic fishery breeding mode (P<0.05). The crude protein and crude fat contents in the PM group were significantly higher than those in the CM group (P<0.05). The photovoltaic fishery breeding mode can promote the E. sinensis growth. The sensory evaluation showed that the hepatopancreas grassy and fatty taste scores in the PM group were higher than those in the CM group. The hepatopancreas' electronic nose response value in the PM group was the highest, and the gonads' grassy odor in the PM group was more noticeable. The principal component analysis (PCA) of the electronic nose showed that the two breeding methods had a significant effect on the hepatopancreas odor profile of female crabs. The determination of volatile compounds by GC-MS showed a total of 44 (PM group) and 33 (CM group) substances identified in the hepatopancreas, mainly including aldehydes (22.59% and 21.83%), alcohols (21.79% and 28.14%), and alkanes (37.03% and 38.00%). In the gonads, 31 (PM group) and 47 (CM group) substances were identified, mostly aldehydes (13.01% and 31.19%), aromatic (7.54% and 3.99%), alcohols (8.18% and 9.69%), and alkanes (67.49% and 44.20%). Twenty-two hexanal-related substances were detected simultaneously in the hepatopancreas and gonads of female crab, while fourteen 2-methyl-2-butenal-like substances were detected only in the hepatopancreas, and fourteen 2-pentenal-like substances were detected only in the gonads. In the hepatopancreas, the total volatiles content in the PM group [(70.53±2.91) ng/g] was significantly higher than that in the CM group [(16.63±0.73) ng/g] (P<0.05), corresponding to 4.2 times more total volatiles in the PM group than in the CM group. Among these, the contents of fatty and grassy flavor substances such as 2-methyl-butenal, 2-methyl-2-butenal, 4-heptenal, 3,5-3,5-octadien-2-one, and 1-octen-3-ol in the PM group were higher than in the CM group (P<0.05). In gonads, the total volatiles content in the PM and CM groups were (68.80±1.86) ng/g and (71.50±4.17) ng/g, respectively, showing no significant difference between the two groups (P>0.05). Nonetheless, the PM group's 2-ethylfuran and D-limonene contents were significantly higher than those in the CM group (P<0.05). The grassy odor in the PM group was stronger in relation to the CM group, and the trimethylamine content in the CM group was 17 times higher than that in the PM group. Sulfur-containing compounds (dimethyl disulfide and thiophene) were detected in the CM group, and the odorous substances in this group were more abundant than those of the PM group, reflecting the inferior odor quality of the CM group. Several different flavor active substances were detected in hepatopancreas (11) and gonads (10) of female crabs under the two breeding methods, respectively. In the hepatopancreas, the OAV of (E)-2-octenal, octenal, (E)-2-nonenal, nonanal, decanal, and 2-pentylpyridine of the PM group was greater than 1, while the CM group's OAV of only (E)-2-nonenal and nonanal was greater than 1. The OAV of (E)-2-nonenal and nonanal was greater than 1 in gonads of the PM group. In the CM gonads, the OAV of hexanal, nonanal, and decanal was greater than 1. Overall, the OAV content was higher than 1 in the hepatopancreas in the PM group, which was higher than that in the CM group. In the gonads, the OAV content was greater than 1 for the CM group, revealing a fishy and ammoniacal odor that resulted in an overall unpleasant odor. PLS analysis showed a strong correlation between the PM group and grassy and fishy odor. The volatiles associated with hepatopancreas in the PM group were octanal, (E)-2-octenal, 2-decanone, 1-octen-3-ol, 2-pentyl furan, among others, while few volatiles associated with hepatopancreas were found in the CM group. Compounds strongly correlated with the CM group gonads included nonanal, hexanal, and other fishy odor aldehydes, trimethylamine, and 2-ethylfuran. The gonads' odor in the CM group was higher than in the PM group. The photovoltaic fishery breeding method can improve the grassy and fatty odors in the hepatopancreas, reduce the content of odor substances in the gonads, and promote the odor quality of female E. sinensis. This study provides a theoretical basis for optimizing and popularizing the photovoltaic fishery breeding method.

Abstract:Vibrio harveyi, Vibrio parahaemolyticus, Vibrio scophthalmi, Vibrio anguillarum, and Photobacterium damselae subsp. damselae are important pathogenic bacteria frequently reported in mariculture animal diseases in recent years. These pathogens exhibit strong pathogenicity, wide epidemic area, and high mortality rates, which usually cause serious economic losses in aquaculture. Strengthening the research on rapid detection technology of these pathogens can help to effectively prevent and control their transmission and infection, and reduce economic losses of the aquaculture industry. Therefore, rapid detection of multiple pathogens in this field can promote the disease control technology development of the aquaculture industry. In this study, the vhhA gene of V. harveyi, toxR gene of V. parahaemolyticus, luxR gene of V. scophthalmi, empA gene of V. anguillarum, and Mcp gene of P. damselae subsp. damselae were selected as the target genes. Specific primers were designed using Primer Premier 5.0 software. V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were used as the target bacteria, V. campbelii and 10 other bacterial species were used as the control group, and sterile water was used as the blank control. The specificity of the primers was verified by the laboratorial conventional real-time quantitative PCR. Thereafter, V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were amplified using the UF-150 microfluidic fluorescence quantitative PCR instrument to verify the feasibility and specificity of the designed primers on microfluidic fluorescence quantitative PCR. The PCR products were digested and recycled, then ligated to PMD-19T vector and transformed into DH-5α competent cells to construct the standard reference material. The extracted plasmid DNA was used as a template for fluorescence quantitative PCR amplification after 10-fold gradient dilution, and a standard curve was drawn. The DNA of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were used to optimize the annealing temperatures for single microfluidics quantitative PCR. The annealing temperatures were set at 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, and 63 ℃, respectively, and the optimal common annealing temperatures of the five strains were finally screened. Meanwhile, the number of reaction cycles was set at 25, 30, 35 and 40, respectively, to verify the optimal number of reaction cycles. The designed specific primers of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were integrated into the microfluidic chip; multiple microfluidic fluorescence quantitative PCR was performed on the integrated chip with the DNA template of the five strains to verify the cross-reaction between the primers. Based on the reaction systems and conditions of single detection methods, the reaction systems and conditions of multiple microfluidic fluorescence quantitative PCR were optimized, and the microfluidic chip was integrated to establish a multiple microfluidic fluorescence quantitative PCR detection technology that could simultaneously detect these five pathogens: V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum and P. damselae subsp. damselae. Scophthalmus maximus, Litopenaeus vannamei, and Apostichopus japonicus were selected as the experimental orgainsms. The five pathogens were mixed with the tissues of S. maximus (muscle, gill and liver), L. vannamei (muscle and gill), and A. japonicus (respiratory tree and intestine). The established multiple microfluidic fluorescence quantitative PCR method was used to detect these tissues which contained the five pathogens, and the conventional real-time fluorescence quantitative PCR was performed for comparison. The results showed that the optimal reaction system of the established detection method was as follows: 2×Taq Pro Universal SYBR qPCR Master Mix 5 μL, primer F/R 1 μL, DNA template 2 μL, and ddH2O 1 μL. The reaction conditions were as follows: 95 ℃ for 30 s, 95 ℃ for 5 s, 61 ℃ for 30 s, and 30 cycles of amplification. The standard curves of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae showed good linearity in the range of 104–109 copies/μL. The linear equations were as follows: y= ‒2.972x+6.73, R2= 0.999; y= ‒3.287x+7.48, R2=0.998; y= ‒3.549x+8.14, R2=0.998; y= ‒3.912x+7.83, R2=0.999; y= ‒3.969x+7.07, R2=0.992, respectively. The method showed strong specificity and high sensitivity to V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae, and the minimum detection limits for these bacteria were 40, 200, 200, 500 and 20 CFU/mL, respectively. The established multiplex microfluidic fluorescence quantitative PCR method was used to detect these pathogens in animal samples and compare the results to those of conventional fluorescence quantitative PCR. The results showed that the accuracy of the established multi-microfluidic fluorescence quantitative PCR method was higher than 96.2% of the conventional real-time fluorescence quantitative PCR method. The average detection time of the samples was only 26 min, which was significantly shorter than that of the conventional real-time fluorescence quantitative PCR reaction time of 1 h 40 min. The multiplex microfluidic fluorescence quantitative PCR method established in this study for the detection of five kinds of mariculture pathogens has strong specificity, high sensitivity, low environmental condition requirements, outstanding portability, and detection accuracy (same as that of conventional real-time fluorescence quantitative PCR), which is suitable for the development of rapid detection technology for aquatic pathogens.

Abstract:Shrimp has become a highly traded global seafood product, with 8 million tons of shrimp produced annually. Acute hepatopancreatic necrosis disease (AHPND) is the most prevalent and severe disease affecting shrimp aquaculture, resulting in considerable economic losses. The AHPND incidence in shrimp farming was as high as 60%–80% in China, resulting in reduced farming capacity and unstable production. Vibrio parahaemolyticus has been identified as the main causative agent of AHPND. In addition, V. harveyi, V. cambelii, V. algolyticus, and V. owenii are capable of causing similar diseases, demonstrating a distinct pathogenic diversity. Previous studies have indicated that not all of the above-mentioned Vibrios species are capable of causing AHPND, and whole gene sequencing and knockout genes have revealed that pirA and pirB are the primary pathogenic factors responsible for AHPND in shrimp. Specifically, the causative agent for AHPND should be a specific strain of Vibrio carrying the binary toxins pirAVp and pirBVp on the extrachromosomal virulence plasmid pVA1. Among them, the pirB toxin mainly determines the pathogenicity of the bacterium, whereas the pirA virulence is relatively weak. Furthermore, it has been demonstrated that the virulence plasmids encoding the binary genes pirAVp and pirBVp are the main causative agents of AHPND. The virulence gene toxR is prevalent in Vibrio and plays an important role through the genetic diversity of 16S rRNA genes during shrimp infection. Real-time fluorescence quantitative PCR technology has less contamination, more accurate quantification, real-time monitoring, and greater automation than conventional PCR technology, which has been utilized in the fields of transgenic detection, environmental science, and medicine. However, this technique is time-consuming, involves multiple instruments and reagents, and requires personnel with extensive professional skills and experience. As a result of its small size, low sample and reagent consumption, rapid detection speed, miniaturization, and integration, microfluidic chip assay technology has emerged as a new focal point in assay technology. Therefore, in this study, we designed specific primers and established a microfluorescence quantitative PCR assay based on two genes, pirA and pirB, to address the genetic similarity of AHPND pathogens carrying a large plasmid encoding a binary toxin, pirA and pirB. The method was specific for the pathogenic pirA and pirB genes, and only when DNA from AHPND-infected samples was tested could the two genes be successfully amplified, while all other pathogenic bacteria were detected with negative results. The sensitivity was high, and the minimum detection limits for the pirA and pirB genes were 5.43×100 and 4.31×101 copies/μL, respectively. Standard curves for pirA and pirB were constructed and demonstrated good linearity in the concentration range of 5.43×109–5.43×104 copies/μL for pirA (y= –3.145x+6.63, R2=0.999) and 4.31×109–4.31×104 copies/μL for pirB (y= –3.015x+5.45, R2=0.999), with an average sample detection time of approximately 26 min. In order to evaluate the efficacy of the method in practice, artificial infection experiments with V. parahaemolyticus were performed. In this study, artificial infection experiments were induced by both injection and immersion, and samples were collected at different time periods to clinically validate the established method and compare its effectiveness in detecting different shrimp tissues, thereby facilitating a more thorough analysis of the pathogenic pathways of infection. The experimental group with injection as the mode of infection was found to be positive for all tissues in all time periods except the water test, which was negative. The experimental group that used immersion as the infection method showed different results for various time periods and with different genetic tests. In terms of the infection method, the tissues could be infiltrated within 2 h using the injection method, whereas the target genes were not detected in the hepatopancreas at 6 h using the immersion method. This indicated that the injection method infiltrated the tissues more rapidly than the immersion method. According to the comparison results of the three genes, pirB was only negative in the intestine at 2 h and positive in all tissues the rest of the time; pirA was negative in the hepatopancreas and intestine at 2 h, only the intestine was negative at 6 h, and all tissues were positive at 12 h; and toxR was negative in all tissues at 2 h. The rate of infestation from rapid to slow showed that pirB > pirA > toxR. Based on the rate of tissue infestation, pirA and pirB were detected in both cheek filaments and muscles at 2 h, making them the most rapid infiltration agents. Therefore, the strategy of using pirB as the primer and gill filament or muscle as the target tissue is more suitable for the rapid detection of AHPND in the field. In this study, we established a method for microfluidic fluorescent quantitative PCR that has the advantages of being rapid, sensitive, high throughput, less contaminated, on-site detectable, and integrated. The method is not only applicable to the laboratory but also meets the requirements of rapid field detection at hatcheries and farms, and can be used as a new technical method for shrimp fry quality detection and disease control.

Abstract:Swimming is of great significance for the survival of fish and directly affects their ability to avoid predators and enemies, hunt and capture prey, carry out mating and reproduction, and migrate. The skeletal muscles of bony fish, which provide power for swimming and account for approximately 40%–60% of the body mass, can be divided into red and white muscle fibers. Red muscle fibers have a slow contraction, strong endurance, high mitochondria content, glycogen, and myoglobin; mainly employ aerobic metabolism; and effectively use oxygen to produce ATP. They are also known as slow-twitch muscle fibers, whose main function in fish is to provide stable and continuous power for the swimming process. White muscle fibers contract quickly but also tire rapidly and mainly use glycolic metabolism. They are also known as fast-twitch muscle fibers and, in fish, provide power for fast swimming behaviors (such as predation and escape). Many studies have indicated a strong correlation between fish swimming habits and the composition of slow and fast-twitch muscles. Most of these studies focused on the correlation between swimming motion and muscle fiber types, but differences in the histological characteristics of fast- and slow-twitch muscle fibers of fish with different swimming habits have rarely been reported. The histological characteristics of muscle fibers include shape, diameter, and density, which are important indicators describing the histological structure of the skeletal muscle in fishes. In this study, we selected three species (Scomber japonicus, Larimichthys crocea, and Paralichthys olivaceus) representing different swimming styles, to clarify the histological characteristics of fast- and slow-twitch muscle fibers, and compared them using hematoxylin-eosin staining of paraffin sections and morphometric methods. The staining showed that the transverse sections of the fast- and slow-twitch skeletal muscle fibers were irregular and the diameter of the fast-twitch muscle fibers was larger than that of the slow-twitch muscle fibers. In S. japonicus, a species engaged in sustained swimming, the fast-twitch muscles were multi-angular, whereas the slow-twitch muscle fibers were multi-columnar. In L. crocea, a species swimming in an extended style, the muscle fibers were long, oval, and had cells with round edges. In P. olivaceus, a species engaged in prolonged swimming, the fast-twitch muscle fibers were oblate and had more connective tissues than the slow-twitch fibers. The slow-twitch muscle fibers of S. japonicus and the fast-twitch muscle fibers of P. olivaceus were finer than the slow-twitch and fast-twitch muscle fibers of these two species, respectively. The longitudinal section of the muscle fibers in the three species were distributed in strips alternating with connective tissue. In addition, the muscle fibers in S. japonicus occupied a larger space and were more loosely arranged than those in the other two species. However, the muscular space between fibers of both types was smaller in P. olivaceus and the muscle cells were more closely arranged. Morphometric results showed that the diameters of fast-twitch muscle fibers were significantly larger than those of slow-twitch muscle fibers (P<0.01). The fast-twitch muscle fibers in S. japonicus were 4.84 times the diameter of slow-twitch muscle fibers, while that ratio was approximately 2.57 in L. crocea and 3.07 in P. olivaceus. The diameter of slow-twitch muscle fibers in L. crocea was the largest among the three species, approximately 1.34 and 1.14 times that of S. japonicus and P. olivaceus, respectively. In contrast, S. japonicus had the largest fast-twitch muscle fiber diameter, approximately 1.41 and 1.35 times that of L. crocea and P. olivaceus, respectively. The slow-twitch muscle fiber density in all three species was significantly greater than that of fast-twitch muscle fibers (P<0.01). The density of slow-twitch muscle fibers was 12.13, 4.60, and 3.91 times than that of fast-twitch fibers, in S. japonicus, L. crocea, and P. olivaceus, respectively. The order of the fast-twitch muscle fiber density was P. olivaceus [(274.60±9.07) unit/mm2] > L. crocea [(205.43±12.63) unit/mm2] > S. japonicus [(118.92±10.74) unit/mm2]. The density of the fast-twitch muscle fiber of P. olivaceus was 2.31 and 1.34 times that of S. japonicus and L. crocea, respectively. The order of slow-twitch muscle fiber density was S. japonicus [(1 442.33±28.25) unit/mm2] > P. olivaceus [(1 073.92±39.40) unit/mm2] > L. crocea [(945.74±19.53) unit/mm2]. Furthermore, the slow-twitch muscle fiber density of S. japonicus was 1.53 and 1.34 times that of L. crocea and P. olivaceus, respectively. The above-described methodology and analysis of differences in the shape, diameter, and density in the skeletal muscle fibers of teleost fish with different swimming habits will provide basic data for further studies on the adaptive evolution and movement physiology of this taxonomic group.