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Abstract:The total saline-alkaline land area in China is approximately 99.1 million hectares, distributed throughout northern China, coastal areas, and areas along the bank of the Yellow River. About 45.9 million hectares of saline-alkaline water areas are distributed around these lands, most of which are athalassic saline water characterized by high pH and high carbonate alkalinity concentrations with various types of ion imbalances. The co-effect of high pH and high carbonate alkalinity would directly lead to the respiratory alkalosis of aquatic organisms. High pH affects the excretion of ammonia, resulting in increased blood ammonia and acid-base imbalance. High ionic coefficient affects the osmotic regulation and breaks the ion balance in aquatic organisms. Thus, saline-alkaline water has not been fully used because of its stressful environmental characteristic. Currently, the lack of suitable objects for the saline-alkaline aquaculture restricts the development of the aquaculture industry on saline-alkaline land. Largemouth bass (Micropterus salmoides) is a potential economic target that has been successfully farmed in some saline-alkaline waters. However, largemouth bass's tolerance range and response mechanism to saline-alkaline water are still unclear. This study evaluated the growth performance of juvenile largemouth bass in a saline-alkaline environment to propose excellent farming species for saline-alkaline aquaculture. First, the juvenile largemouth bass response to 48 h carbonate alkalinity and 96 h semi-lethal salinity was determined. Hereafter, the saltwater group [SW, salinity of 7.50±0.07, and carbonate alkalinity of (1.81±0.12) mmol/L], alkaline water group [AW, salinity of 0.35±0.02, and carbonate alkalinity of (9.96±0.03) mmol/L], and freshwater control group [FW, salinity of 0.13±0.01, and carbonate alkalinity of (1.82±0.11) mmol/L] were set to comparatively study the growth parameters, physiology parameters, and muscle texture characteristic indexes of largemouth bass under long-term saline-alkaline stress. For the growth experiment, largemouth basses were acclimated to and reared in FW, SW, and AW conditions for 105 days. Triplicate of 30 individuals each were set for each condition using an experimental plastic tank with 100 L of water. Each fish's body length and weight were measured every 15 days after being anesthetized with MS-222. For the physiology parameters study, five largemouth basses were randomly selected from each group at the end of the growth experiment. The fish were anesthetized with MS-222 to draw 20 μL of blood from the tail vein using a syringe moistened with lithium heparin, which was immediately centrifugated to measure osmolality. Another 30 largemouth basses were taken and subjected to 24 h carbonate alkalinity stress. The experimental conditions were the same as AW group, and the control group was the same as FW group. During the stress period, feeding was stopped, and blood was drawn from five randomly selected fish every 6 h and centrifuged immediately to determine blood ammonia (blood ammonia kit A086-1-1 by Nanjing Jiancheng). Plasma osmolality was measured using an osmometer (Wescor Vapro 5520 Vapor Pressure Osmometer, USA). For the muscle texture characteristic index study, five largemouth basses were randomly selected from each group at the end of the growth experiment. After being anesthetized with MS-222, the muscles on the fish's backside (3.01±0.14) g were taken by using a surgical scalpel and scissors, the muscles' outer skin was cut off, and the sampled muscle sizes were standardized to (2.04±0.12) cm3. After sampling, the TMS-Pro texture analyzer (Food Technology Corporation, USA) was used to measure the muscle texture characteristics, employing the TPA mode, test speed of 30 mm/min, deformation amount of 50%, and return distance of 30 mm. The results showed that in the 48 h carbonate alkalinity group, the semi-lethal concentration was (29.92± 3.90) mmol/L, while the fish could survive safely in water with salinity below 10 mmol/L. After 105 days of farming, there are no significant differences in the survival rate and final weight among different groups, in which the specific growth rate (SGR) showed a regular variation. During 15~45 days and 60~75 days, SGR decreased continuously, while it increased during 45~60 days and 75~90 days. The condition factors of the largemouth bass were less than 3 in all groups, with an increase from FW to AW and from AW to SW groups. In the AW group, the blood ammonia within 24 h showed an increase, then a decreased, and finally stabilized. In the SW group, the osmolality was (319.53±29.51) mOsm/kg, lower than the (300.00±16.44) mOsm/kg observed for the FW group. Largemouth bass raised in saline-alkaline water had better texture characteristics. Largemouth bass raised in SW group had a higher muscle hardness of (34.70±4.86) N, while a higher springiness of (1.06±0.10) mm was observed in the AW group. In summary, the largemouth bass could adapt to the relatively high saline-alkaline environment and be cultured in typical saline-alkaline water with pH from 8.84 to 8.89, carbonate alkalinity from 9.89 to 10.31 mmol/L, salinity from 6.68 to 7.21, showing good muscle quality characteristics with high muscle hardness and springiness. The success of largemouth bass in saline-alkaline water aquaculture has provided an opportunity to promote the aquaculture of this fish in a saline-alkaline stressful environment, providing the theoretical basis for the mechanisms involved in this adaptation process. Our study will broaden the scope of aquaculture in saline-alkaline water, improving the economic benefits and providing the basic parameters for the quality evaluation of fish in saline-alkaline fisheries.

Abstract:There are approximately 64 million hm2 of low-lying saline-alkali water resources in inland China, accounting for 55% of the total lake area. The characteristics of saline-alkali water are: high salt content, high alkalinity (high pH), and complex ion composition. Most freshwater fish cannot survive or reproduce in these conditions. To utilize saline-alkali water resources for aquaculture, researchers have introduced salt-tolerant fish, such as Luciobarbus capito, Chalcalburnus chalcoides aralensis, Leuciscus waleckii, and Oreochromis niloticus. In the initial stage of aquaculture, fish fry are not yet adapted to saline-alkali conditions because their body structure and physiological functions are not underdeveloped. The survival rate of juvenile fish is low because of the stress response to saline-alkali water. Studies of saline-alkali aquaculture have shown that proper acclimation can effectively improve the survival rate, growth, antioxidant, and immune properties of fish. L. capito belongs to the Cyprinidae family (subfamily Barbinae). It occurs mainly in the Caspian Sea and the Aral Sea of Western Asia. L. capito is an economically important fish, with adults weighing 5~ 10 kg. The species has excellent breeding characteristics, high saline-alkali tolerance, varied food habits, fast growth, delicious meat, and strong stress resistance. In 2003, it was introduced from Uzbekistan to China. To date, research has reported on L. capito artificial breeding technology and breeding biology but has not yet considered salt-alkali domestication. To study the physiological adaptability of L. capito to alkalinity with regards to blood physiology, biochemistry, and antioxidative stress, we used juvenile L. capito (13.66±1.26) g to test NaHCO3 alkalinity acclimation. The control group was cultivated in freshwater. The acclimation group was subjected to 20 mmol/L alkalinity for 7 d and then placed in 40 mmol/L alkaline water. The non-acclimation group was placed in 40 mmol/L alkaline water. The experimental media were prepared with tap water and NaHCO3 (domestic, analytically pure) after aeration for 2 d, and the alkalinity was measured and calibrated by acid-base titration. The fish were fed once a day and fasted for 24 h before sampling. Blood physiological and biochemical indices (water osmotic pressure, white blood cells, lymphocytes, neutrophils, monocytes, hemoglobin, red blood cells, platelets, platelet hematocrit, urea, and albumin), as well as indices related to the liver antioxidant system of juvenile fish in 40 mmol/L alkalinity water, were measured at 0 h, 6 h, 12 h, 24 h, 48 h, 96 h, and 7 d. In the control group, the same sampling was conducted at the corresponding moments. The physiological and biochemical indices for both domesticated and non-domesticated fish initially increased and then decreased across the sampling period (P<0.05). In both the domesticated and non-domesticated groups, the antioxidant indices of fish liver tissue, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA), increased initially and then decreased across the sampling period (P<0.05). The peak values of each parameter in the domesticated group were significantly lower than those of the non-domesticated group (P<0.05). No significant changes were observed in the control group during this period (P>0.05). The peak values of leukocytes, lymphocytes, neutrophils, and monocytes appeared at 48 h in the acclimated group and at 24 h in the non-acclimated group; the blood hemoglobin content, red blood cell content, platelet content, hematocrit, SOD, and CAT peaked in both the acclimated and non-acclimated groups at 24 h. After 7 d, the urea, lymphocyte, monocyte, platelet, red blood cell, hemoglobin, and albumin content of the blood and aspartate aminotransferase (AST), alanine aminotransferase (ALT), SOD, CAT, GSH-PX, and MDA content of the liver tissue were significantly lower in the acclimation group than in the non-acclimation group (P<0.05), but the plasma albumin content and glutathione catalase in the acclimated group returned to the level of the control group (P>0.05). Studies have shown that after alkalinity acclimation, juvenile L. capito are less stressed and the body tissue shows less damage and greater recovery under high alkalinity. At the physiological level, the body has higher adaptability. This study provides a theoretical basis for the establishment of saline-alkali aquaculture technology for the cultivation of L. capito that is based on the adaptability of the species to gradual changes in the alkalinity of the environment.

Abstract:The total saline-alkaline land area in China is approximately 99.13 million hectares, distributed throughout northern China, coastal areas, and areas along the Huanghe River. About 45.87 million hectares of saline-alkaline water areas are spread around these lands, most of which are athalassic waters characterized by a high pH value above 8.8, associated with high-carbonate alkalinity and various types of ions imbalances. The saline-alkaline land and water cannot be directly used for agriculture, and most of them are arid. The development of aquaculture in saline-alkaline land is not only beneficial to expanding the aquaculture area but also can restore the saline-alkaline soil, which is of great significance to food security and ecological restoration. Saline-alkaline aquaculture is one of the main inland aquaculture models developed in the past ten years. With the maturity of aquaculture technologies, the saline-alkaline aquaculture area has expanded year by year, which has brought earnings to local farmers. China has abundant saline-alkaline water resources. The high pH and high-carbonate alkalinity of these waters restrict the survival, growth, and reproduction of aquatic animals. Litopenaeus vannamei is highly resistant to stress and has a certain tolerance to saline-alkaline water. Under short-term saline-alkaline stress, the expression of the carbonic anhydrase (CA), Na+/K+-ATPase, and other ion-regulated genes of L. vannamei were induced, and the acid-base and osmolality balance were determined by strengthening ion regulation. At present, relatively few studies on gene regulation of L. vannamei under long-term stress have been performed. Although L. vannamei farming has been successful in saline-alkaline water, the survival rate is unstable, and there are few reports on the selective breeding of L. vannamei tolerant to salinity and alkalinity. Through independent innovation, a family-based "multi-trait compound breeding technology for aquatic animals" has been established in China. These techniques have laid a good foundation for developing improved L. vannamei strains. To effectively utilize saline-alkaline water resources, it is urgent to conduct L. vannamei salt-alkali-tolerant breeding and promote the healthy development of the saline-alkaline aquaculture industry. L. vannamei has strong environmental adaptability and relatively high tolerance to saline-alkaline water. It is one of the main species of saline-alkaline aquaculture. However, its survival rate in high pH and high-alkaline environments is not stable. To explore the response mechanism to long-term high-alkaline stress, L. vannamei was exposed to low-alkaline water as the control group (LSW, carbonate alkalinity of 3 mmol/L, salinity of 6, pH of 8.1) and to high-alkaline stress (AW, carbonate alkalinity of 10 mmol/L, salinity of 6, pH of 8.8) for 42 days. The intestine and gill of L. vannamei raised for 42 days were used as the experimental materials. Transcriptome sequencing was performed using the Illumina platform. After splicing analysis and gene annotation, the differentially expressed genes and regulatory pathways regulated under high-alkaline stress were screened and analyzed, with further verification by qRT-PCR. The results showed 243 differentially expressed genes in both tissues, of which 98 were up-regulated and 145 were down-regulated. The differentially expressed genes in the intestine were enriched for glucose metabolism, carbohydrate digestion and absorption, bile secretion, ABC transmembrane transport, and tight junction related pathways. The differentially expressed genes in gills were enriched for glutathione metabolism, bicarbonate transport, arginine synthesis, sugar metabolism, and ion transport related pathways. The ten most significant differentially expressed genes were further studied and verified by qRT-PCR. Carbonic anhydrase (CA1, CA10), ecdysone-inducible protein (Eip74EF), and β-galactosyltransferase (UGT8) genes in gills were down-regulated. However, the expression of Na+/K+-ATPase-α (NKA-α), Na+/K+ transporting ATPase interacting (NKAIN), ammonia transporter (Rhbg), and malate dehydrogenase (MDH) were up-regulated under high-alkaline stress. The transcriptome expression pattern and qRT-PCR results were consistent. We speculated that these genes may be involved in the shrimp stress response to high-alkaline stress. L. vannamei showed a relatively strong high-alkaline tolerance, which may be compensated by down-regulating the expression of CA to prevent alkalosis, up-regulating Rhbg to prevent ammonia accumulation and NKA-related genes to maintain the osmotic balance. The ecdysone function was probably affected as the Eip74EF gene was down-regulated. This study provides basic data for further analyzing the physiological response mechanisms of L. vannamei under long-term highly alkaline stress.

Abstract:The global levels of soil and water salinization and alkalinization are increasing with the influence of climate and topography changes, as well as other natural and human factors. Saline-alkaline water (SAW) all over the world has specific characteristics such as high alkalinity, high pH, and complex water quality types, which inhibits the survival and culture of common aquatic animals. The ridgetail white prawn Exopalaemon carinicauda is an economically important marine shrimp with many advantages, such as widely environmental adaptability, rapid growth, and good reproductive capability. It is potentially suitable for large-scale culture in SAW; however, its adaptability to this environment remains unclear. Exploring the E. carinicauda adaptability mechanism to SAW will help to guide culture management for marine crustaceans. In this study, the DNA methylomes of the E. carinicauda gill tissue cultured in SAW and normal seawater (SW) were analyzed and the impact on gene regulation was investigated by MethylRAD sequencing. The results showed 2 347 003 and 416 176 methylations at the CG and CWG sites (W = A or T), respectively. Comparing the SAW and SW groups, the CG and CWG loci in the SAW group increased slightly, indicating that SAW induced more DNA methylation in the gill cells that activated or inhibited pathways and played a crucial role in the environmental changes adaption. Methylation was prevalent in the exon, intron, splice site, and upstream and downstream regions of the E. carinicauda gill genes, as well as in the intergenic regions. DNA methylation sites were mostly distributed in the Genebody. The DNA methylation distribution curve peaked in the downstream sequence of the transcription start site and upstream sequence of the transcription termination site. The methylation label frequency was significantly higher in these regions in relation to other sequences. A total of 8805 differential methylation sites (DMSs) were screened, including 8189 CG DMSs and 616 CWG DMSs. Obviously, the CG DMS was significantly higher than the CWG DMS. The intergenic and intron regions accounted for a large proportion of the DMS observed. Overall, the DMS showed a higher trend in the genic downstream regions of the gene relative to upstream regions. The Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) based on the DMS showed enrichment of genes involved in the "development, heterochrony, and protein disables isomerase activity," which played a role in the CG level down-regulation. In addition, "incubation involved in sorocarp development" and "nucleus and double-stranded RNA binding" were molecular functions up-regulated by the CG methylations. The down-regulated genes with CWG methylation were enriched for the "regulation of transcription and DNA template" process, while the up-regulated genes were enriched for the "epithelial cell migration and open trail” system. These two processes were induced in E. carinicauda cultured in saline-alkali water. The key genes in the HIF-1 signal and spliceosome pathways, such as hif-p, hk, and sf3b1, play an important role in response to saline-alkaline stress; therefore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis based on DMS of the DEG was performed. In addition, 158 CG methylated genes (MGs) were detected in DEGs from shrimps exposed to SAW, among which 77 and 81 were up- and down-regulated, respectively. Moreover, 94 CWG MGs were differentially expressed, from which 33 and 61 were up- and down-regulated, respectively. GO enrichment analysis of the CG MGs showed significant enrichment of the "muscle organ development" process; CWG MGs were significantly enriched for the "vesicle-m transport" and "membrane mediated zinc binding plasma transport" processes. This indicated that E. carinicauda was damaged by the saline-alkaline environment, but the shrimp may adapt to this stress by adjusting the ion balance. The KEGG enrichment analysis indicated that lipid metabolism and signal transduction pathways may play crucial roles in the E. carinicauda gill tissue response to saline-alkaline stress. Moreover, changes in lipid peroxidation and physiological metabolism may be caused by long-term saline-alkaline stress. The energy metabolism pathway was significantly enriched and many different genes in the lipid metabolism pathway were expressed in the stressed E. carinicauda. Therefore, it was speculated that the changes in DNA methylation level might play an important role in response to saline-alkaline stress. Overall, the results showed that a series of E. carinicauda physiological activities related to environmental adaptation was activated by SAW. In addition, a small number of methylated loci were negatively correlated with gene expressions, which indicated a complex relationship between DNA methylation and gene regulation. Although ADAM17, FPC-l, and CYP450 showed hypomethylation, the corresponding differentially MGs showed a significantly increased expression level in a saline-alkaline environment. Therefore, genomic DNA methylation may promote gene expression under saline-alkaline stress, including the ADAM17, FPC-l, and CYP450 genes. This study analyzed the DNA methylation levels in gill tissue of E. carinicauda cultured with SAW and provided information that will further elucidate the molecular mechanisms involved in crustacean adaptation to saline-alkaline environment.

Abstract:Aquaporins are a family of cell membrane proteins, and their key role is to specifically transport water molecules and other neutral metabolic molecules. Moreover, aquaporins play an important role in regulating the balance between internal and external osmotic pressure in organisms. Here, aquaporin 4 and 11 genes from Exopalaemon carinicauda were successfully cloned using rapid amplification of cDNA ends cloning. The open reading frame of EcAQP4 is 621 bp, encoding 206 amino acids, with a predicted molecular weight of 21.673 kDa and a theoretical isoelectric point of 8.30; it is a hydrophobic protein with five transmembrane structural domains. The open reading frame of EcAQP11 is 783 bp, encoding 260 amino acids, with a predicted molecular weight of 28.490 kDa and a theoretical isoelectric point of 5.40; it is a hydrophobic protein with four transmembrane domains. In sequence alignment, AQP4 in E. carinicauda shared the highest homology with that in Macrobrachium rosenbergii (94.63%), while AQP11 in E. carinicauda shared the highest homology with that in Penaeus monodon (81.47%). Furthermore, RNA interference was used for silencing EcAQP4 and EcAQP11 expression to verify their function. In carbonate alkalinity stress, the mortality of E. carinicauda increased significantly following RNA interference. At 72 h, mortality reached 45% and 55% in the EcAQP4 and EcAQP11 groups, respectively, being significantly higher than that in the control group. The blood osmotic pressure of E. carinicauda in the EcAQP4 group was significantly higher than that in the control group. Similarly, the blood osmotic pressure of E. carinicauda in the EcAQP11 group was significantly increased. In summary, aquaporins play important roles in regulating osmotic pressure and maintaining ion balance in response to alkalinity stress in E. carinicauda.

Abstract:Mitten crab (Eriocheir sensu stricto) is an important aquaculture species in China, with three main species: Chinese mitten crab (Eriocheir sinensis), Japanese mitten crab (Eriocheir japonicus), and Hepu mitten crab (Eriocheir hepuensis). Mitten crab has a catadromous life cycle. Paired mitten crabs mate and spawn in estuaries, and the progeny migrate upriver into freshwater rivers to grow. Mitten crab is widely distributed in major basins along the coast of China. Wild mitten crab populations are distributed from the Suifenhe River in the Heilongjiang Province in the north to the Nanliujiang River in the Guangxi Zhuang Autonomous Region in the south. Suifenhe mitten crab is an indigenous wild mitten crab population distributed in the Suifenhe River basin of Dongning City, Mudanjiang City, Heilongjiang Province. It is a highly salt-tolerant aquatic animal, and the current mitten crab germplasm is severely degraded. Therefore, the development of a new mitten crab germplasm is of great significance for crab industry development and salt-alkali aquaculture in China. In September 2020 and 2021, ground cages were used to capture 299 wild crabs (156 females and 143 males) from the Suifenhe River basin of Heilongjiang to investigate the parameters of sexual maturity, body weight (BW) profile, color, total edible yield, and proximate composition. The sexual maturity of wild Suifenhe mitten crab was determined by puberty molting, and BW was measured using an electronic balance. The hepatosomatic index (HSI, %) and gonadosomatic index (GSI, %) were calculated from their respective weights and the ratio of BW. A high-precision spectrophotometer was used to determine the color of the wet and dry samples. Vacuum freeze-drying was used to determine the moisture content of the edible tissues (hepatopancreas, gonads, and muscles). Protein, lipid, and ash were analyzed by the AOAC and Soxhlet extractor method. In late September, wild crabs in the Suifenhe River basin (Dongning Section) in Heilongjiang Province comprised of two stages: immature and sexually mature. The ratio of immature females was significantly lower than that of sexually mature ones (P<0.05); however, it was the opposite for males. Regardless of sex, the average BW of sexually mature individuals was significantly higher than that of immature individuals (P<0.05). The average BW of mature females was (110.51±2.42) g, and the average BW of mature individuals was (147.79±5.94) g. The average BW of sexually mature female crabs was mainly concentrated in the range of 75.00~149.99 g, and the percentage of mature female individuals (≥100.00 g/ind) was 55.94%. The average BW distribution of sexually mature male crabs was relatively scattered, and the percentage of mature male individuals (≥125.00 g/ind) was 60.00%. On September 29, the HSI and GSI of females were significantly higher than those of males, whereas the rates of muscle yield (MY) and condition factor (CF) were higher in males than those in females (P<0.05). The brightness (L*) and yellowness (b*) values of the female carapace were significantly lower than those of the males (P<0.05). The crude protein content in female gonads and muscles was significantly higher than that in male carapace, whereas the moisture content in male gonads was significantly higher than that in females (P<0.05). The results of this study provide basic data for the development and utilization of Suifenhe mitten crab germplasm resources. Wild adult Suifenhe mitten crab had larger BW and a higher percentage of large-sized individuals than pond-reared E. sinensis in the north of China. The proportion of edible tissues, condition factor, color, and proximate composition of Suifenhe mitten crab were not significantly different from those of E. sinensis from the Liao River and Yangtze River. Meanwhile, the ratio of crude protein to crude lipid in ovaries and muscles was in accordance with the human body’s need for high-protein and low-fat foods. Hence, Suifenhe mitten crab is a new crab germplasm with significant development potential. These results provide basic data for the development and utilization of Suifenhe mitten crab germplasm resources.

Abstract:To establish the caudal fin cell line of Schizothorax biddulphi, this study employed the tissue block method with fetal bovine serum (FBS) DME/F12 medium for in vitro cultivation of the tail fin, using previously established tail-fin cell lines (BICF1) of S. biddulphi. In this study, the effect of NaCl salinity and NaHCO3 alkalinity on the proliferation of tail fin cells was investigated using the MTT method. The main results are as follows: the BICF1 suspension culture was propagated to 45 generations. The optimum medium was DME/F12. The optimal FBS concentration was determined to be 20%. The optimum temperature was 25℃. The population doubling time of the 10th generation BICF1 was 28.11 h, showing an "S" type growth. The 6th generation BICF1 in liquid nitrogen was recovered after freezing for 180 days. Trypan blue staining showed that after recovery, (87.85±0.66)% of BICF1 cells were active, and the cells could proliferate and pass to next generation. The BICF1 cell line is free of contamination by bacteria, fungi, and mycoplasma. The sequencing results of mitochondrial 16S rRNA of the 10th generation BICF1 were consistent with the GenBank gene sequence, and the consistency rate of BICF1 and JQ844133.1 was 100%, proving that BICF1 was from S. biddulphi. BICF1 cell proliferation increased with salinity of 1, 2, 4, and 6, but decreased with salinity of 6, 8, and 10; BICF1 cell proliferation was the highest at 6. The NaHCO3 alkalinity of BICF1 increased at 2, 3, 4, 5, and 6 g/L, whereas the proliferation of BICF1 cells decreased at 6, 7, 8, and 10 g/L, respectively. BICF1 cell proliferation was the highest at 6 g/L. Cell proliferation first increased and then decreased with increasing salinity and alkalinity. This study aimed to provide a scientific basis for the rational development and utilization of genetic resources of S. biddulphi, including the establishment and protection of the germplasm.

Abstract:Antarctic krill (Euphausia superba) is a vital part of the food web, supporting substantial fishery in the Southern Ocean. Krill represent the world´s largest animal protein pool, rich in lipids, nutritional long-chain polyunsaturated fatty acids, mineral elements, and carotene, with great potential for development. It is of great significance to understand the changes in fishing grounds, as they profoundly affect the krill population. The distribution characteristics of Antarctic krill fishing grounds in 48 areas were analyzed based on the production statistics of Antarctic krill fishing vessels in China from 2010 to 2019. The gravity center migration trajectory model and standard deviational ellipse (SDE) model were used to analyze the trends and characteristics of Antarctic krill fishing grounds. The results showed that the production of Antarctic krill was mainly concentrated in sub-region 48.1, accounting for 70.30% of total production. The yields of the 48.2 and 48.3 sub-regions showed little difference, accounting for 14.28% and 15.42%, respectively. The annual catch per unit effort (CPUE) curve increased over time, with a minimum value in 2012 and a maximum value in 2019. The monthly CPUE first increased and then decreased, with the minimum value in January and the maximum value in June. The annual and monthly gravity center of Antarctic krill in sub-region 48.1 moved southwest. The annual gravity center of Antarctic krill in sub-region 48.2 moved to the east, but the range of movement was small, and the monthly trends were weak. The annual gravity center of Antarctic krill in sub-region 48.3 moved to the south, and the monthly gravity center moved to the northwest. According to SDE analysis, krill fishing grounds in sub-region 48.1 had the widest distribution and the greatest dispersion, with the strongest directivity and the most obvious centripetal force in sub-region 48.3. The center of gravity of the fishing ground was distributed along the Bransfield Strait in sub-region 48.1, east of the South Orkney Islands in sub-region 48.2, and northeast of the South Georgia Islands in sub-region 48.3. Clustering results showed that the annual fishing ground gravity centers were concentrated in sub-region 48.1. The annual fishing ground gravity centers of sub-regions 48.2 and 48.3 were relatively concentrated in all years, except in 2017.

Abstract:To understand the status of early-stage fish resources in the Yibin Section in the upper reaches of the Yangtze River and guide the protection of fish resources in this section, surveys were carried out from April to July in the years of 2017~2019. The results showed that there were 22 species of eggs and larvae in five families and three orders, and the largest proportion was in the Cyprinidae. Among these, 13 species produced drifting eggs, and six species were endemic to the upper reaches of the Yangtze River. The breeding time of different fish had obvious annual differences, but the peak of fish breeding occurred in June. From 2017 to 2019, the number of drifting eggs was estimated to be 12.10×106, 70.42×106, and 35.77×106 for each year, respectively. The number of larvae was estimated to be 30.95×106, 41.47×106, and 39.44×106, respectively. The number of eggs of endemic fish in the upper reaches of the Yangtze River was estimated at 15.91×106 over three years. According to the calculation, there were many spawning sites of fish drifting upstream of the Yibin Section, and the larger spawning sites were mainly located in the reaches of Zhouba, Peachwan, and Hualong Wharf. The spawning sites of Rhinogobio typus were distributed from Dongyuemiao to Xiangjiaba, with the largest cumulative spawning amount in three years, up to 40.93×106. The spawning grounds of R. typus tended to migrate downstream. The spawning grounds of Ctenopharyngodon idellus are mainly distributed in the Mamingxi Wharf and Peachwan areas. The results of the canonical correlation analysis showed that environmental factors such as water level, water temperature, and water discharge had different effects on the density of drifting fish eggs. Lepturichthys fimbriata and Parabotia fasciata lay eggs when the water temperature and water discharge are high. The reproductive conditions of C. idellus and Leptobotia microphthalma were similar to each other, and they were highly correlated with the water level, water discharge, and velocity. The spawning of R. typus and Coreius heterokon was significantly affected by changes in dissolved oxygen and transparency. Although affected by many factors, such as the development of cascade hydropower in the Jinsha River, the Yibin Section, as an important part of the mainstream national nature reserve of rare and endemic fish in the upper Yangtze River, is still a crucial distribution area for the spawning and breeding of many fish species, and the fish populations have slowly recovered in recent years. Therefore, we should strengthen the early resource research in this section, carry out habitat conservation and remediation, and implement a “10-year Fishing Ban” to promote resource recovery.

Abstract:With extensive utilization of fishery resources, it is important to understand the related feeding habits of large marine predatory fish. In traditional studies on stomach contents, cephalopod organisms in the stomach of fish are identified mainly on the basis of cephalopod body characteristics. However, because the physical characteristics of cephalopods are affected by digestive juices in fish stomachs, the appearance of cephalopods are easily destroyed. Therefore, there are some limitations in the identification of cephalopods based on these characteristics. Cephalopod beaks have morphological characteristics that can be used for species identification and classification, but the classification of cephalopod beaks from stomach contents of large marine predatory fish has not yet been conducted. Thus, in order to investigate the possibility of classifying cephalopod beaks from stomach contents of six large marine predatory fishes, we examined a total of 36 fish stomachs from the South Atlantic, Middle East Pacific, and Western Indian Oceans in October 2017, March~May 2018, and November 2018, and analyzed cephalopod beak morphology. In this study, two-dimensional digital images of beak morphology were collected. The side and top morphologies of each lower beak were collected for species identification. Based on the morphological characteristics of the beak and the terminology related to beak morphology, a classification retrieval table for beak morphology was developed. The morphology coefficients were analyzed by clustering at the family level by extracting the elliptic Fourier descriptor coefficients of beak morphology. Results showed that a total of 17 species of cephalopods were found in two orders and ten families through species identification using beak morphology. Analysis of the beak morphology from stomach contents revealed that the morphological characteristics of the beaks of 17 species of cephalopods are clearly different among species. According to the classification retrieval of beak morphology, the lower beak of the rostrum of 13 species of Teuthoidea was triangular and isosceles triangle, and the lateral wall was approximately rhomboid. The wings were well-developed. The lower beak of the rostrum of four species of Octopoda was developed, and the lateral wall was approximately a long quadrangle. Wing development occurred. The morphology of beaks in different cephalopods has unique characteristics, and the characteristics of each species are very different, as indicated during the analysis. Cluster analysis of morphological information from beaks was effective for distinguishing cephalopods at the order level. In the cluster analysis, beak morphology was divided into two main parts: one mainly corresponded to Teuthoidea and the other corresponded to Octopoda. The identification and classification of beak morphology from stomach contents can be used as a marker for the biological identification of cephalopods. This not only can support the recognition of cephalopods from stomach contents, but also enrich the identification content of cephalopods from stomach contents. This study lays the foundation for the classification and identification of cephalopods in the stomachs of large marine predatory fish.

Abstract:Trophic level, a basic and measurable feature in complex food networks, represents the position of organisms in the ecosystem and reflects the vertical structure of the food web. The study of trophic levels is used to interpret sophisticated ecological relationships through simplified functional groups and is of great significance to the construction of ecosystem models and the study of nutrient cycling. The construction of the Three Gorges Dam and the operation of the reservoir have fragmented the river ecosystem. The influences of reservoir storage and operation vary between river sections with disparate geomorphic characteristics. Environmental heterogeneity may cause temporal and spatial variation in the structure of the fish community of each section of the mainstream from the reservoir. After two decades, research on fish trophic levels has mainly focused on the initial stage of storage and operation of the reservoir, while information on the fish trophic level in the mainstream remains scarce. To evaluate the long-term spatiotemporal variation in fish trophic levels in the Three Gorges Reservoir, nitrogen stable isotopes of 38 fish species and three snail species were collected from the Mudong, Fuling, Yunyang, and Zigui river sections of the main stream of the Three Gorges Reservoir from July 2018 to January 2019, and the continuous spectra of fish species trophic levels in the reservoir were constructed based on stable isotope analysis. The results showed that the average δ15N value of snails was 8.37‰, ranging from 8.29‰ to 8.53‰, and there was no significant spatial difference between the river sections. Except for the Mudong section, the δ15N values of snails in the other river sections were significantly higher in the flood season than in the dry season. Nitrogen stable isotope analysis of fish samples showed that the average value ranged from 5.31‰ to 17.79‰, with an average of 11.02‰. Taking snails as the baseline organism, fish trophic levels were calculated for each river section according to season, and the results showed that the average trophic level of fish species in the reservoir area was 2.67, ranging from 1.47 to 4.12. There was only one fish species with a trophic level higher than 4; 12 species of fish (31.6%) with trophic levels between 3 and 4, mainly piscivorous and benthivorous fish; 19 species of fish (50.0%) with trophic levels between 2 and 3, mainly omnivorous, benthivorous, and planktivorous fish; and six species of fish (15.8%) with trophic levels between 1 and 2, including three herbivorous and three omnivorous fish species. The trophic levels of fish combinations with different feeding habits varied, and were found to be piscivorous > benthivorous > planktivorous fish and omnivorous > herbivorous fish, indicating that fish feeding preference was one of the key factors determining the trophic level of fish. Between different river sections, there were differences in the range of fish trophic levels, food chain length, and trophic levels of fish combinations with the same feeding habits. However, there was no significant spatial difference in the trophic level of the fish community among the river sections. A paired sample t-test showed that there was no significant difference in fish trophic levels between the flood and dry seasons. Within the same river section, the seasonal fluctuation in food chain length decreased with distance from the dam. Compared with the initial stage of reservoir impoundment, the fish trophic level increased significantly, and the community structure and trophic characteristics of high trophic-grade fish changed to a certain extent. In particular, Coilia brachygnathus with higher trophic levels gradually expanded and became the dominant species in the mainstream of the reservoir area, which might affect the trophic structure of fish in the greater Three Gorges Reservoir area. The results of this study provide a reference for the assessment, protection, and rational utilization of fishery resources in the Three Gorges Reservoir area.

Abstract:In this study, a correlation analysis of 57 single nucleotide polymorphism (SNP) markers and growth-related traits of Leiocassis longirostris was conducted using 115 samples under the same growth conditions. The results showed that 10 loci were related to growth-related traits. Among them, three (cluster-65_137265, cluster-65_111833, and cluster-65_137642) loci had a significant influence on growth-related traits (body weight, total length, body length, and head height) (P<0.05). Cluster-65_120392, cluster-65_105077, and cluster-65_5592_0 had significant influences on body weight, total length, and body length (P<0.05). Cluster-65_110382 significantly influenced total length, body length, and head height (P<0.05). Cluster-65_19497 and cluster-24304_1 had a significant influence on the total length and body length (P<0.05). Cluster-65_130153 significantly influenced body length and head height (P<0.05). We also estimated the genetic diversity parameters for the 10 loci. The mean observed heterozygosity, expected heterozygosity, and the polymorphism information content (PIC) were 0.537, 0.467, and 0.357, respectively. In addition, cluster-65_137265, cluster-65_111833, cluster-65_110382, and cluster-65_137642 were associated with anion exchange protein 2-like isoform X1, Netrin-4-like, Tec isoform X2, anion exchange protein 2-like isoform X1, and carbamoyl-phosphate synthase [ammonia] have the highest similarity, respectively. This finding indicates that these four genes may be involved in the regulation of L. longirostris growth. The study provides primary data for the genetic improvement and selective breeding of L. longirostris.

Abstract:Granzyme B is an indispensable mediator of the immune inflammatory response, which can induce apoptosis of target cells by activating caspase 3. In this study, we cloned the full-length cDNA of Cynoglossus semilaevis granzyme B-like gene (CsGzmBl) and analyzed its sequence characteristics and expression levels. The full-length cDNA of CsGzmBl was 923 bp, containing a 49 bp 5ʹUTR, 94 bp 3ʹUTR, and 780 bp ORF regions encoding 259 amino acid proteins with 19 amino acid signal peptides. The genome structure of CsGzmBl is highly conserved and consists of five exons and four introns. The CsGzmBl protein possesses two N-terminal glycosylation sites, a catalytic triad “His63Asp112Ser207,” a “PHSRPYMA” domain, and six conserved cysteines. qRT-PCR indicated that CsGzmBl was expressed in different tissues of healthy C. semilaevis adults, with the highest expression in the spleen, followed by that in the head kidney, kidney, liver, and gills, with the lowest expression in muscle. After infection with Vibrio harveyi, CsGzmBl was up-regulated to varying degrees in six immune-related tissues (spleen, intestine, liver, skin, gills, and kidneys) at different time points compared to the control group at 0 h. These results indicate that CsGzmBl plays an important role in the immune response of C. semilaevis against V. harveyi infection.

Abstract:Galectin-6 is a member of the β-galactoside-binding lectin family, which has been widely studied in mammals (e.g., Lgals6). Galectin-6 has been cloned from several teleost fishes, such as Maylandia zebra, Esox lucius, and Poecilia formosa, but there are few studies on its function. In this study, we analyzed the molecular characteristics of Galectin-6 extracted from Paralichthys olivaceus (PoGalectin-6) and studied its immune-related functions. The full length of the open reading frame (ORF) of PoGalectin-6 cDNA is 1089 bp, encoding 362 amino acids containing two carbohydrate recognition domains (CRDs). Multiple sequence alignment and phylogenetic tree analysis showed that PoGalectin-6 is highly similar to Scophthalmus maximus Galectin-4 (approximately 80.9%). Tissue distribution experiments demonstrated that PoGalectin-6 is specifically expressed in intestinal tissues. The expression level of PoGalectin-6 in the intestine significantly increased after Edwardsiella tarda stimulation, and the highest expression level was observed at 12 h after infection. Recombinant PoGalectin-6 (rPoGalectin-6) exhibited binding ability to gram-positive (Bacillus subtilis, and B. cereus) and gram-negative (Aeromonas salmonicida, E. tarda, and Vibrio vulnificus) bacteria. rPoGalectin-6 showed Ca2+-dependent agglutination activity against B. pumilus, B. subtilis, A. salmonicida, and E. tarda. This study suggests that PoGalectin-6 may play an important role in the immune response against E. tarda infection, laying the foundation for exploring the immune functions of Galectin-6 in teleosts.

Abstract:A 12-week feeding experiment was conducted to evaluate the effects of fish oil replacement by mixed microalgae on growth performance, body composition, digestive enzyme activities, and antioxidant indices in the intestinal tract of juvenile turbot (Scophthalmus maximus) [mean initial weight (39.69±0.25) g]. Four isonitrogenous and isoenergetic diets were formulated with graded contents of dietary fish oil replacement by mixed microalgae, 0, 25%, 50%, and 100%, designated D1, D2, D3, and D4, respectively. The results showed that weight growth rate (WGR) and feed efficiency (FE) decreased with an increasing replacement ratio; no significant differences were found between D1, D2, and D3 groups (P>0.05), but all three had significantly higher values than the D4 group (P<0.05). The D1 group showed the highest value of the hepatosomatic index (HSI), which was significantly higher than that in other groups (P<0.05). With an increasing replacement ratio, the body and muscle crude lipid contents increased at first, and then decreased, being significantly higher in the D4 group than in the D1 group (P<0.05). The C20:4n-6 and n-6 PUFA in the whole fish body reached their peaks in the D4 group (P<0.05), with significantly higher values than those in the D1 group (P<0.05). However, EPA, DHA, and n-3 PUFA significantly decreased in D4 (P<0.05). The activities of lipase and trypsin in the intestinal tract reached their peaks in the D3 group. The activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and catalase (CAT) in the intestinal tract of the D3 group were significantly higher than those in the D1 group (P<0.05). Additionally, the activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LZM), as well as the complement component 3 (C3) contents in the intestinal tract, showed the same tendency as SOD. The malondialdehyde (MDA) contents in the intestinal tract were not significantly different across the groups (P>0.05). These results indicate that 50% fish oil replacement by mixed microalgae does not influence the growth performance of juvenile turbot, and may increase the digestive enzyme activities and antioxidant ability in the intestinal tract.

Abstract:Exopalaemon carinicauda is one of the special economic shrimp in China, which has become an important object of pond single breeding and mixed breeding in recent years. The ridgetail white prawn has good breeding prospects, however, due to its small size and relatively small spawn, it currently mainly relies on natural seedlings, catching natural seedlings or placing broodstock to propagate their own seed, which severely restricts the development of large-scale artificial aquaculture. To achieve large-scale artificial breeding of white shrimp, we must break through the difficulty of full artificial breeding, and the bait is the nutritional basis to ensure the normal growth and development of seedlings. Biological diet generally refers to various aquatic plants, animals and microorganisms that can be artificially cultivated and suitable for aquatic animals to eat. Compared with traditional feed, biological food has many advantages, such as easy to cultivate, fast reproduction, rich nutrition, and strong palatability, and can effectively enhance the disease resistance ability of breeding objects. At present, the commonly used biological food includes Chaetoceros muelleri, chlorella, microcytococcus, cyanobacteria, rotifer, Artemia, cladocera, copepod and amphipoda. In this study, we analyzed the survival rate, larval stage index, and digestive enzymes of Exopalaemon carinicauda larvae (Z1~P) fed live food, including group A (feeding only on Artemia nauplii), group B (Chaetoceros muelleri and A. nauplii mixed feeding), group C (Brachionus plicatilis and A. nauplii mixed feeding), and group D (C. muelleri, B. plicatilis, and A. nauplii mixed feeding). The results showed that group A had the highest metamorphic rate, and all individuals metamorphosed into larvae within 14 days, while the other three groups took 15 days. The survival rates of larvae metamorphosed into larval shrimp were 84.33%, 84.67%, and 83.00% for groups A, B, and C, respectively, and the differences among the three groups were not significant (P>0.05). A large number of larvae during the Z5~P stage died in group D, and the survival rate was only 35.67%. Moreover, the activities of pepsin (1.94 U/mg prot), lipase (2.35 U/mg prot) and α-amylase (0.13 U/mg prot) were the highest in group A, while the activities of pepsin (0.08 U/mg prot), lipase (0.91 U/mg prot) and α-amylase (0.08 U/mg prot) were the lowest in group D. The activities of α-amylase in groups B and C were 0.12. The activities of lipase (1.78 U/mg prot) and respectively (0.39 U/mg prot) in group C were 0.35 U/mg prot and 0.04 U/mg prot higher than those in group B. This study suggests that feeding E. carinicauda larvae with A. nauplii (3~5 ind./mL) is expected to increase the survival rate of larvae and accelerate metamorphosis during the nursery phases and supplementation with C. muelleri can alleviate the rate of deterioration in aquaculture water.

Abstract:After 40 days of exposure to different concentrations of nitrate at a salinity of 5, the effect of nitrite on the immune system and growth performance of Litopenaeus vannamei was explored to provide a theoretical basis for constraining the effect of nitrite subacute stress on the species. Five nitrite concentration groups (0.50, 0.90, 1.70, 3.20, and 6.00 mg/L) and a control group (0.05 mg/L) were used in this experiment. Furthermore, we detected the activity of immune-related enzymes, malondialdehyde (MDA) content, and relative expression of immune- and growth-related genes. Results showed that the mortality rate of L. vannamei increased with an increasing nitrite concentration, and the weight gain rate (WGR) and length gain rate (LGR) in the 6.00 mg/L group were significantly lower than those in the control group (P<0.05). The activity of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), and alkaline phosphatase (AKP) in the serum and hepatopancreas increased under the influence of nitrite in 0.90 and 3.20 mg/L groups. The activity of SOD in the hepatopancreas was significantly higher than those in the control group when the concentration was higher than 0.50 mg/L (P<0.05). CAT activities in the 0.50, 0.90, and 1.70 mg/L groups were significantly higher than those in the control group. The activity of SOD and CAT in the serum first decreased, then increased, and then decreased again with an increasing concentration of nitrite. The activities of AKP and ACP in the serum and hepatopancreas in the 0.90 mg/L group were significantly higher than those in the control group (P<0.05). There was no obvious change in MDA content across groups. In addition, serum glutamic-pyruvic transaminase (GPT) activity was significantly higher in the serum than in the control group (P<0.05). The results of quantitative real-time PCR showed that the relative expression of mn-sod and hsp70 genes in the hepatopancreas was significantly higher than that in the control group, except in the 0.50 mg/L concentration group (P<0.05). The relative expression of cat, trx, tgase, trypsin, and chitinase genes in the hepatopancreas was significantly lower than that in the control group (P<0.05). After 40 days of challenge with different concentrations of nitrite, the growth and immune function of L. vannamei clearly diminished. The concentration of nitrite should be controlled below 0.50 mg/L in the production process at a salinity of 5 to ensure the healthy farming of L. vannamei.

Abstract:In this study, the retention efficiency (RE) of Meretrix meretrix, Mercenaria mercenaria, and Ruditapes philippinarum were measured by adding sediment or unicellular algae to adjust the concentration of suspended particles. The results showed that in natural seawater, the RE of M. meretrix, M. mercenaria, and R. philippinarum reached maximum retention when the particle sizes were 8 μm, 6 μm, and 6 μm, which were 51.1%, 59.6%, and 62.6%, respectively. With an increase in particle number concentration, the minimum particle sizes for maximal retention in M. meretrix shifted to approximately 9 μm, 13 μm, and 14 μm in low (4.32×107 cells/L), mid (5.27×107 cells/L) and high (6.65×107 cells/L) particle number concentration, respectively. The relative standardized RE decreased to 49.7%, 33.4%, and 26.2%. As in M. meretrix, the minimum particle sizes for maximal retention in R. philippinarum shifted from 6 μm to 9 μm, 12 μm, and 14 μm with the change in particle number concentration, but the change in maximal retention was not significant. The change in maximal retention in M. mercenaria was not significant; however, the minimum particle sizes for maximal retention shifted to approximately 8 μm, 9 μm, and 10 μm, respectively. With the increase in particle concentration, the change in maximal retention and minimum particle size for maximal retention was not significant in three different particle concentration, low (5.7 mg/L), mid (11.8 mg/L) and high (23.3 mg/L), retention of M. mercenaria changed significantly, 60.7%, 27.6%, and 25.5%, respectively. The results indicate that the RE of M. meretrix and R. philippinarum were more sensitive to particle concentration, while the RE of M. mercenaria was more sensitive to organic content.

Abstract:To analyze the nutritional composition and quality of Tectus pyramis in Mirs Bay and Qionghai in Hainan, the basic nutritional composition, amino acid composition, fatty acid composition, and some mineral elements in their muscle tissues were determined by the national standard method. Amino acid score, chemical score, and the essential amino acid index were used to evaluate the amino acids. The results showed that the ash content of the muscle of T. pyramis caught in Qionghai was significantly higher than that caught from Mirs Bay, but there was no significant difference in other basic nutrients (P>0.05). Total amino acid contents in the muscle tissue for individuals caught from Mirs Bay and Qionghai in Hainan were (18.45±0.13) g/100 g and (18.83±0.03) g/100 g, respectively; in both groups, the essential amino acid content was more than 27%, branched-chain amino acid content was more than 13%, and delicious amino acid content was 55%. The content of polyunsaturated fatty acids in the muscles was more than 48%. The contents of linoleic acid, α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in Mirs Bay were significantly higher than those in Qionghai, and the DHA+EPA content was as high as 11.79%. Among the mineral elements detected, the content of calcium and trace element iron was the highest in the two kinds of T. pyramis, followed by trace element zinc. The calcium and zinc contents in the muscles of T. pyramis in Qionghai were significantly higher than those in Mirs Bay. In conclusion, T. pyramis have a high protein content, rich polyunsaturated fatty acids, and high muscle nutritional value, with a great potential for development. These data provide a reference for the further development and utilization of new snail food in island and reef waters.

Abstract:Aeromonas hydrophila is one of the main pathogens seriously endangering mandarin fish farming and production. In order to explore the changes in host gene expression levels in A. hydrophila infection, screen for immune-related genes, and analyze the molecular mechanism of Siniperca chuatsi infected with Aeromonas hydrophila in response to pathogenic bacterial infection. The head kidney tissues of the infected and control groups were collected 24 h after inoculation and used for mRNA extraction, and transcriptome sequencing was performed using high-throughput sequencing technology. A total of 53 040 single genes (unigenes) were obtained from the original sequencing data through de novo assembly. The results of gene differential expression analysis showed that there were 526 differentially expressed genes, of which 254 were upregulated and 272 were down-regulated in the infected and control groups. Among the differentially expressed genes, several key genes involved in the immune response, such as interleukin-8, interleukin-6 receptor, interleukin 17 receptor, lysozyme, complement receptor type 1, antigen processing and presentation, serine protease inhibitor, and E3 ubiquitin-protein ligase were present. Gene ontology analysis revealed that the different genes were mainly involved in immune response and inflammation. A KEGG analysis showed that there were 89 significantly enriched and immune-related metabolic pathways mainly involved with endocytosis and phagosomes. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) verification was performed on seven DEGs, and the results showed that RT-qPCR was consistent with RNA-Seq analysis. These results lay a theoretical foundation for a follow-up study of gene function and in-depth exploration of the molecular defense mechanism of S. chuatsi against pathogenic microorganism infection.

Abstract:Enterocytozoon hepatopenaei (EHP) has caused serious losses in shrimp aquaculture in China in recent years. For cultured shrimp, disease prevention cannot be carried out by vaccination because of the lack of specific immune mechanisms. The most effective measure to prevent disease outbreaks is to conduct on-site rapid pathogen detection as far as possible for relatives or seedlings or to carry out on-site detection early in the onset of shrimp, timely detection, and identification of pathogens. There is, therefore, an urgent demand for rapid detection techniques and kits for controlling and preventing EHP. A novel, highly sensitive kit, developed in our laboratory, can achieve rapid detection of EHP in the field by optimizing the three steps of tissue nucleic acid extraction, nucleic acid amplification, and nucleic acid detection, and provide a practical solution for early rapid screening and detection of shrimp EHP disease. To validate the newly developed highly sensitive kit for rapid EHP detection in the field, a systematic evaluation of the six performance parameters of the kit was carried out in this study. The analytical specificity results showed that the kit did not cross-react with the DNA/RNA extracted from healthy shrimp or shrimp infected with five other pathogens, including WSSV, CMNV, SHIV, VpAHPND, and IHHNV. The analytical sensitivity analysis showed that the detection limit was 101 copies/μL with shrimp DNA preparation. The diagnostic sensitivity and diagnostic specificity were determined to be 91.7% and 99.2%, respectively, in tests of 298 clinical samples, in comparison with the TaqMan qPCR protocol of EHP. The repeatability was 100% for strongly positive and negative samples and 95.8% for weakly positive samples. The period of validity of the kit was tested and found to be over 7 months at –20℃ storage and over 12 months at –40℃. The study results demonstrated that the kit is a simple, sensitive, specific, and accurate tool for the rapid detection of EHP in practical applications.

Abstract:The aim of this study was to explore the immunoprotective mechanism and protective effects of oral specific egg yolk immunoglobulin (IgY) against white spot syndrome virus (WSSV) infection in Pacific white shrimp (Litopenaeus vannamei). Different doses of WSSV IgY agent (0, 0.2%, and 0.5%) were added to shrimp feeds and administered to juvenile L. vannamei for 28 days. The survival rate of juveniles was measured within 14 days of artificial infection with WSSV. The activity of immune enzymes and the relative expression levels of immune genes in shrimp hepatopancreas were measured after 3 days of WSSV infection. The results showed that compared with the control group without IgY, the 0.2% IgY agent group showed significantly higher enzyme activity by superoxide dismutase (SOD) and phenoloxidase (PO), and significantly lower enzyme activity by acid phosphatase (ACP) and alkaline phosphatase (AKP). The relative expression level of heat shock protein 70 gene (Hsp70) also significantly increased, while the relative expression levels of lectin gene (lectin) and β-1,3-glucan binding protein-lipoprotein gene (β-GBP-HDL) significantly decreased in the 0.2% IgY agent group. SOD activity significantly increased while PO activity did not change significantly, while ACP and AKP activities decreased in the 0.5% IgY agent group. The relative expression level of Hsp70 also significantly increased, and the relative expression level of lectin did not significantly change, while the relative expression level of β-GBP-HDL significantly decreased in the 0.5% IgY agent group. The artificial infection results showed that after 14 days of WSSV infection, the survival rates of shrimp in the 0.2% and 0.5% IgY agent groups, and the control group were 48.89%, 87.78%, and 0, respectively. The survival rate of shrimp in the 0.2% and 0.5% IgY agent groups was significantly higher than that in the control group, and the survival rate of shrimp in the 0.5% IgY agent group was also significantly higher than that in the 0.2 % IgY agent group. Specific IgY agents can change the course of a disease, delay disease onset and death of immune shrimp, and significantly improve the survival rate of shrimp over a certain period. The results showed that oral specific IgY agents could affect the activity of immune enzymes and the expression level of immune genes in the hepatopancreas of L. vannamei, significantly improving their ability to resist WSSV infection. This study provides a basis for the application of IgY agents in the prevention and control of WSSV infection, and also provides a reference for research on the anti-WSSV infection mechanism of IgY agents.

Abstract:In this study, peptide PGRP (designated HdPGRP) was identified and characterized from the abalone Haliotis discus hannai. Multiple alignments and phylogenetic analyses strongly suggested that HdPGRP is a new member of the PGRP superfamily and belongs to the short PGRP family, similar to peptides from other marine mollusks. The full length of HdPGRP is 1467 bp, encoding a polypeptide of 354 amino acids (aa) with a signal peptide (1~18 aa), an SH3b domain (93~160 aa), a typical PGRP domain (179~322 aa), and an Ami_2 domain (191~322 aa). In addition, four conserved Zn2+-binding sites (H209, Y255, H318, and C330) and five conserved amide-catalysis sites (H209, Y255, H318, T328, and C330) were found in the HdPGRP sequence. In abalone, hdpgrp exhibited different tissue expression patterns, and was strongly expressed in the hepatopancreas, moderately expressed in hemocytes, mantle, and gills, and slightly expressed in muscle. Vibrio anguillarum is one of the main pathogens of H. discus hannai; after V. anguillarum infection, expression of hdpgrp in hemocytes showed a trend of first increasing and then decreasing, reaching a maximum at 24 h. Subsequently, expression of HdPGRP decreased, and there was no significant difference compared with the control group at 72 h, demonstrating that expression of HdPGRP had returned to normal levels. SDS-PAGE results showed that recombinant HdPGRP (rHdPGRP) has a molecular mass of 30 kDa, which is in line with the value predicted for HdPGRP. PGRPs usually have amidase activity, degrading peptidoglycan by hydrolyzing the amide bond that links peptide units to muramic acid residues of glycan strands. rHdPGRP exhibited Zn2+-dependent amidase activity and catalyzed the degradation of insoluble peptidoglycan. In addition, rHdPGRP exhibited significant antibacterial activity against the gram-positive bacterium Micrococcus luteus in the logarithmic phase in the presence of Zn2+, indicating that the antibacterial activity of HdPGRP might be dependent on amidase activity. In summary, HdPGRP plays an important role in PGRP-mediated antibacterial mechanisms, especially for eliminating invading bacteria.