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Abstract:Species composition, ecological role, diversity, and clustering characteristics of fish species were analyzed by means of the species similarity, diversity index, and redundancy analysis (RDA) fishery resource surveys data collected in November 2015 (autumn) and May 2016 (spring) in the Zhoushan coastal fishing ground. The results showed that a total of 81 fish species were identified, which belonged to 63 genera in 39 families of 12 orders, with the bulk belonging to offshore, warm-water species. The Jaccard similarity index is 37.04% at the middle dissimilarity level. Analysis showed that the dominant species in the spring was Amblychaeturichthys hexanema, which accounted for 42.16% of the total catches. The dominant species in the autumn was Harpodon nehereus, which accounted for 43.56% of the total catches. The diversity index value of spring was higher than in autumn. However, the two seasonal diversity indices had similar planar distribution. Diversity was observed to be the lowest in the southern region of the survey area increasing toward the north, and a similar trend was seen from east to west in the survey region. The analysis of similarities (ANOSIM) test showed that the differences between the communities were highly significant. The similarity percentage (SIMPER) showed that seven species of fish, including Decapterus maruadsi, Collichthys lucidus, and H. nehereus, were typical species belonging to different groups in the spring. Fish community structure differed in autumn compared to spring primarily in that there were nine species of fish, including Sardinella nymphaea, Engraulis japonicus, and Benthosema pterotum. The results of the RDA indicated that the main environmental factors influencing the structure of fish community in the Zhoushan coastal fishing grounds were salinity and temperature.

Abstract:The immune system of teleost, similar to mammals, is divided into two subsystems: Innate immune system and acquired immune system. Acting as the first line of defense against invading pathogens and pathogenicity factors, the innate immune system can be activated by the pattern recognition receptor (PRRs) which can recognize the pathogen-associated molecular pattern (PAMPs) and activate the downstream inflammatory response accordingly. MAPK(mitogen-activated protein kinase) signaling pathway is an important signal transduction system in eukaryote cells, which can mediate extracellular signaling into the cell and participate in various cellular processes such as cell proliferation, apoptosis, and immune response. JNK (c-Jun N-terminal kinase) signal pathway is an important branch of MAPK signal pathway. JNK have been extensively studied in mammals, but not adequately in the teleost. In this research, we designed experiments to study the role of JNK2 genes in the antimicrobial reaction of Japanese flounder and their effects on the downstream of the pathway in order to provide a theoretical basis for the study of JNK signaling pathway in fish. In this study, the JNK2 gene sequences of Japanese flounder were obtained from the transcription database of flounder and named after PoJNK2. The PoJNK2 ORF region encodes is 1263 bp and encodes 420 amino acids. The domains of PoJNK2 genes were predicted by SMART server. The results showed that the JNK2 gene had a typical serine-threonine protein kinase domain S_TKc. The expression levels of PoJNK2 genes in different tissues of flounder were analyzed by the qRT-PCR technique. It was found that PoJNK2 were widely expressed in the immune tissue of Japanese flounder. Therefore, it was preliminarily speculated that PoJNK2 played a major role in the immune response of Japanese flounder. We designed an infection experiment of Edwardsiella tarda in vivo and immune stimulatory cell experiments in vitro to investigate the role of PoJNK2 in the immune response of Japanese flounder. The expression of PoJNK2 was up-regulated in the spleen, kidney, and gill of Japanese flounder after injecting E. tarda in vivo. At the same time, the gill cells of flounder (FG) were stimulated by E. tarda, Poly I:C and TNF-α. The expression of PoJNK2 was up-regulated at different time points. In addition, we overexpressed PoJNK2 in FG by cell transfection and it was found that PoJNK2 had significant regulatory effects on the regulation of downstream pro-inflammatory cytokines and transcription factor AP-1. The role of PoJNK2 in regulating the immune response of Japanese flounder was further clarified.

Abstract:Frozen sperm from Epinephelus lanceolatus and eggs from E. bruneus and E. moara were used for artificial insemination. The metamorphosis development and growth of larval juvenile of the two hybrid offspring, Helong grouper (E. bruneus♀×♂E. lanceolatus) and Yunlong grouper (E. moara♀×♂E. lanceolatus), were observed and compared. The results showed that the rates of fertilization of the two hybrids were similar, but the malformation rate in the Helong grouper was higher than that of Yunlong grouper, while the hatching rate was lower than that of the Yunlong grouper. The post embryonic development of hybrid offspring was divided into larval stage, juvenile stage and young fish stage based on the features of the yolk-sac, the second dorsal fin spine and the first pelvic fin spine, scale and body color. At temperature of 24~26℃ and salinity of 29~31, DO≥10 mg/L, the yolk sac of Helong grouper and Yunlong grouper disappeared 5 days after hatching (dah). At 41 dah, these two species of fish transited to the juvenile stage at the same time, and the lengths of the second dorsal fin spine and the pelvic fin spine were (9.74±0.42) mm and (7.79±0.39) mm, and (9.59±0.46) mm and (5.81±0.09) mm, respectively, reaching the longest value, and then began to shorten. At 51 dah, Helong grouper juvenile fish completed metamorphosis and transited to the young stage with a total length of (25.46±1.16) mm. The time of Yunlong grouper transited to the young stage was later than Helong grouper (56 dah), with the total length of (23.24±1.21) mm. At 62 dah, there were four brown patches on the body surface of the Helong grouper, and six brown patches on the body surface of the Yunlong grouper. The growth rate of the Helong grouper was greater than that of the Yunlong grouper, and the whole length of the Helong grouper was 1.04 times as long as that of the Yunlong grouper at 70 dah; the Helong grouper showed a faster growing heterosis than the Yunlong grouper. The daily growth formula of the full length of the Helong grouper was L=2.6208e0.0465x, R²=0.9603 and that of the Yunlong grouper was L=2.7158e0.0453x, R²= 0.9451. The relationship between preanal distance and days for the Helong grouper was P=1.3371e0.0483x, R²=0.9643 and that of the Yunlong grouper was P=1.4017e0.0467x, R²=0.944. At 1 dah, the daily growth rates of the full length Helong groupers and Yunlong groupers reached the maximum, were 27.89% and 30.03%, respectively. At 13 dah, the daily growth rate of preanal distance of the brown Helong grouper was the maximum (25.21%), and that of Yunlong grouper reached a maximum (27.01%) at 12 dah. The results of this study showed that the Helong grouper expressed faster heterosis than the Yunlong grouper, and provided abundant biological data for the growth and breeding of crossbred offspring from the same father but a different mother grouper.

Abstract:Coilia nasus taihuensis has become a dominant species of fish in Taihu Lake, China. However, C. nasus taihuensis is often confused with C. brachygnathus, and identification problems still exist today. The fish with length ratio of supermaxilla/head > 1 are believed to be the former species, while those with length ratio of supermaxilla/head < 1 are believed to be the latter. As previous studies have classified all of the Coilia fish in Taihu Lake as C. nasus taihuensis, there are doubts about the accuracy of the results of these studies on genetic differentiation between C. nasus taihuensis and C. brachygnathus. In light of the above information, we have used morphological features to distinguish between C. nasus taihuensis and C. brachygnathus, determined genetic divergences among the two putative species, and tested whether the Cyt-b sequence can identify unknown individuals at early life stages between C. brachygnathus and C. nasus taihuensis. In the present study, we have determined the genetic diversity, genetic structure, genetic divergence, and population demographic history between the two phenotypes of C. nasus taihuensis and C. brachygnathus, using the mitochondrial Cyt-b gene. A total of 22 haplotypes were found among 47 sequences of the Cyt-b gene, from the 33 individuals of C. nasus taihuensis and 14 individuals of C. brachygnathus. The results showed that 26 polymorphic nucleotide sites were detected among all of the Cyt-b sequences, including 12 parsimony-informative sites. The value of Kimura’s two-parameter distance for C. nasus taihuensis and C. brachygnathus was 0.002 and 0.001, respectively. Moreover, the value of Kimura’s two-parameter distance among C. nasus taihuensis and C. brachygnathus, was 0.005. This situation means that the genetic divergence among the two putative species was higher than that within each species. Fst analysis showed a significantly higher level of genetic variance among the two putative species (Fst=0.703, P≤0.001). The gene flow between the two putative species was 0.212, lower than 1, and suggesting that the two phenotypes had almost no gene exchange. Additionally, two distinct clades were detected by phylogenetic analysis, and they were for C. nasus taihuensis and C. brachygnathus, respectively. A haplotype was shared by these phenotypes. Furthermore, the median-joining network produced was similar to the topology of the phylogenetic tree. In summary, mtDNA analysis of this study revealed that there were significant genetic divergences between C. nasus taihuensis and C. brachygnathus, the gene flow between them was weak, and two distinct clades could be detected by phylogenetic analysis, and the Cyt-b sequence could be used to differentiate at early life stages between C. brachygnathus and C. nasus taihuensis.

Abstract:Salinity is an important environmental factor affecting the growth and metabolism of fish. The gill, kidney, and intestine are the main osmoregulatory organs involved in the adaptation of fish to changes in environmental salinity, and aquaporins (AQPs), the cystic fibrosis transmembrane regulator (CFTR), and the Na+/H+ exchanger (NHE) are important osmoregulatory genes in these organs. To study the osmoregulatory function of AQP1, AQP3, CFTR, and NHE1 in turbots (Scophthalmus maximus) under low salinity stress, their expressions in the gill, kidney, and intestine of turbots at salinity levels of 5 and 10 (5- and 10-salinity groups, respectively) were detected by quantitative real-time PCR. The results showed that the expression of AQP1 was very low in the gill but high in the kidney and intestine. Under low salinity stress, the expression of AQP1 in the gill did not change significantly in either of the salinity groups, but it increased significantly in the kidney and intestine (P<0.05). The expression of AQP3 was very low in the kidney, high in the gill, and low in the intestine. Under low salinity stress, the expression of AQP3 in the kidney did not change significantly in either of the salinity groups, but it increased significantly in the gill and intestine (P<0.05). Similarly, the expression of CFTR was very low in the kidney, high in the gill, and low in the intestine. Under low salinity stress, the expression of CFTR in the kidney did not change significantly in either of the salinity groups, but it decreased significantly in the gill and intestine (P<0.05). The expression of NHE1 was low in the gill and intestine but high in the kidney. Under low salinity stress, the expression of NHE1 in the gill did not change significantly in either of the salinity groups, but it increased significantly in the kidney and intestine (P<0.05). These results indicate that the expressions of the four genes vary according to tissue type, salinity, and time, thus reflecting their functional specificity. Under low salinity stress, these genes responded positively and their expressions changed to varying degrees, thus suggesting the roles of AQP1, AQP3, CFTR, and NHE1 in the adaptation of turbots to low salt environments. In addition, the results of this study can provide theoretical basis for brackish water aquaculture and desalination aquaculture of turbots as well as theoretical and technical support to improve the breeding of turbot varieties and their adaptation to low salinity environments.

Abstract:Human activities and environmental pollution have caused varying degrees of damage to Paramisgurnus dabryanus resources in China. To explore the genetic differentiation and phylogenetic relationship of P. dabryanus populations in China’s Pearl River Basin, eight microsatellite markers were used to analyze the genetic diversity of nine geographical populations (Foshan, Gaoyao, Fengkai, Zhaoqing, Ruyuan, Lechang, Shaoguan, Heyuan, and Huizhou) of P. dabryanus in the Pearl River Basin. The analysis detected 69 alleles in eight microsatellite loci. The average number of alleles (Na) and effective alleles (Ne) per locus were 8.6 and 4.0, respectively, and the average observed heterozygosity (Ho) and expected heterozygosity (He) were 0.4426 and 0.7030, respectively. The average genetic differentiation coefficient (Fst) and number of migrants per generation (Nm) of the nine P. dabryanus populations were 0.2452 and 0.7697, respectively, which indicates that genetic differentiation among populations was high and genetic communication was low. Cluster analysis was performed by the unweighted pair group method with arithmetic mean (UPGMA) method, based on the genetic distance among nine populations. The resulting UPGMA tree was divided into two main branches. The Shaoguan, Foshan, and Ruyuan populations were monophyletic and formed one clade. The remaining six P. dabryanus populations formed the other branch, which contained three smaller branches, which were composed of the Lechang and Zhaoqing populations, the Heyuan and Huizhou populations, the Gaoyao and Fengkai populations. These results suggest that the genetic diversity of nine P. dabryanus populations in the Pearl River Basin was high, and there was a certain genetic differentiation among the populations, which could be used for further breeding.

Abstract:Sinibrama taeniatus, a cyprinidae fish belonging to Cultrinae, inhabits only the upper reaches of the Yangtze River, mainly in the Sichuan Province. Due to habitat loss associated with the Three Gorges Project and overfishing, sharp decreases in S. taeniatus populations have been forecast. Surveys of larval and juvenile S. taeniatus were conducted to accumulate developmental data to perfect large-scale artificial breeding technologies and provide a theoretical basis for conservation and recovery of this endemic species. Morphological and anatomic development of larval and juvenile S. taeniatus were with a digital camera system. Newly hatched larvae measured (4.54±0.04) mm at (25.0±0.5)℃. The volume of yolk sacs was (0.26±0.01) mm3, the anterior portion of which was oval and the posterior portion of which was rod-shaped. The development of yolk sac larvae from hatching to yolk absorption took approximately 8 days and the special growth rate of total length was 5.99%. During that period, larvae started feeding on the third day and the mixed nutrition stage lasted 5 days. The relationship between yolk sac volume (V) and age (D) can be expressed as V = −0.0049 D3 + 0.0369 D2 − 0.1333 D + 0.2583 (R2＝0.9947). Late larvae with disappeared yolk sacs completely relied on exogenous material to obtain energy until scales appeared. This developmental stage lasted 25 days and the special growth rate of total length was 2.16%. During the juvenile period, scale development lasted 53 days, and the special growth rate of total length was 0.90%. The relation between total length (TL) and D of larval and juvenile S. taeniatus can be described by TL = −7 × 10−7 D3 −0.0011 D2 + 0.2820 D + 4.7083 (R2＝0.9957).

Abstract:Using a reduced-representation genome sequencing (2b-RAD) technique, this study performed genome-wide single-nucleotide polymorphism (SNP) screening of 649 individuals selected from three consecutive breeding generations (G9~G11) in a population of Fenneropenaeus chinensis named “Huanghai 2”. Genetic structure and genetic diversity of the parents’ population were further analyzed using these markers. In total, 66985 SNPs were developed and genotyped in the 649 shrimp. A genetic analysis showed that the average nucleotide diversities (Pi) were 0.1439, 0.1587, and 0.1674 in G9, G10, and G11, respectively. The average observed heterozygosities (Ho) were 0.1388, 0.1515, and 0.1609, respectively. The polymorphic information contents (PIC) were 0.1241, 0.1360, and 0.1430. Genetic diversity parameters of the G9~G11 parent population showed an undisputed upward trend, although it was not significant. An F-test showed that the total Fst value of all three generations was 0.0061, and the degree of genetic differentiation between adjacent generations was weak (G9~G10 0.0029, G10~G11 0.0026). Genetic distances of adjacent generations decreased slightly, to 0.0029 and 0.0026 in G9~G10 and G10~G11, respectively. Gene flow was 62.91~94.63 in all generations, which indicated that sufficient gene exchange. This study confirmed a certain impact of artificial selection on genetic diversity and genetic structure in F. chinensis breeding populations. It also showed that current breeding strategies (selection pressures of 4% to 5% in each generation), do not undermine genetic diversity in parental populations. Genetic differentiation in each generation’s breeding population was small, and genetic structures tended to be stable. The study provides a basic database for genetic analysis of F. chinensis at the molecular level, and theoretical guidance and data support for the formulation of a F. chinensis breeding program.

Abstract:Tachypleus tridentatus is a rare marine living fossil with immense economic and scientific research value. However, recently, over fishing and indiscriminate killing have led to a sharp decline in the number of horseshoe crabs. Artificial release proliferation is not only an important way to recover horseshoe crab populations, but also to protect biodiversity and promote the sustainable development of fishery. We conducted a preliminary study on artificial breeding of horseshoe crabs T. tridentatus in Zhanjiang, Guangdong Province from May to September 2018. When the water temperature was 28℃~ 32℃, salinity was 26~30, and pH was 7.4~7.8, 9 pairs of T. tridentatus laid 87130 eggs with a fertilization rate of 56%~68%. After 45 days, the embryos hatched into the first instars of juvenile T. tridentatus. The hatching rate was 92%, and 48268 T. tridentatus larvae were obtained. To study the correlation between phenotypic traits and body weight, five quantitative traits (carapace length X1, carapace width X2, carapace height X3, abdomen length X4, and body weight Y) of the first instars of T. tridentatus, 500 individuals were measured, and subjected to correlation analysis, multiple regression analysis, path analysis, and determination coefficient analysis. The results showed that there were considerable correlations (P<0.01) among the five measured traits. Path coefficients of various morphological traits for body mass reached a highly significant level (P<0.01), and the largest path coefficient was used in analyzing the width of the head and chest armor. This indicated that the width of the head and chest armor had the greatest direct effect on body mass. The trend of determination coefficient analysis was comparable to that of path analysis. The elevated total determination coefficient (Σd=0.863) between phenotypic features and body weight indicated that the selected traits were practically useful. The multiple regression equation for estimating body weight was Y=‒0.228+0.248X1+0.073X2+0.333X3+0.073X4. It indicates that the carapace width of the first instars of T. tridentatus has the greatest influence on body mass. Therefore, the width of cephalothorax should be the first choice in germplasm evaluation and seedling screening.

Abstract:In this study, the apoptotic protein activator 1 (apoptotic protease activating factor-1, Apaf-1) gene of the swimming crab (Portunus trituberculatus) was cloned using the RACE technique. The full length of the gene was 2032 bp, and the ORF was 1050 bp, encoding 349 amino acids, with a predicted molecular weight of 39.13 kDa, and a theoretical isoelectric point of 7.67. The homology and phylogenetic analysis showed that the homology of Apaf-1 with Litopenaeus vannamei Apaf-1 was 51.27%, and it was also clustered with L. vannamei. Tissue expression analysis showed that the relative expression level of the Apaf-1 gene was highest in the hepatopancreas, followed by the muscle, heart, gill, and eyestalk, and was lowest in the hemocytes and epidermis. After different salinity stresses, the expression level of Apaf-1 in the stage Ⅱ juvenile crab reached its maximum at 3 h, and then decreased and then increased. Apaf-1 showed an upward trend in the gills after salinity 20 stress after 80 d, and an upward and downward trend in the hepatopancreas, suggesting that the change of salinity could affect the expression of Apaf-1 in the gill and hepatopancreas. The results showed that the expression of Apaf-1 reached its peak at 48 h after injection of Vibrio parahaemolyticus, which was 2.76 times higher than that of the control group (P<0.05), and increased 12 h after the injection of white spot syndrome virus (WSSV), which was 1.25 times higher than that of the control group (P<0.05). In the hepatopancreas, the expression of Apaf-1 reached its peak at 72 h after the injection of Vibrio parahaemolyticus, which was 5.44 times higher than that of the control group (P<0.05), and 12 h after the injection of WSSV. The peak value was 5.89 times higher than that of the control group (P<0.05), and the expression was up-regulated as a whole. This study provides a reference for further understanding the physiological function of the Apaf-1 gene in Portunus trituberculatus.

Abstract:Simulation and Validation of Manila Clam Ruditapes philippinarum Growth with a DEB-Based Individual Growth Model in Jiaozhou Bay

Abstract:Sepioteuthis lessoniana is a common species of cephalopods. In order to understand the nutritive composition of its muscle, we measured and analyzed the general nutrients; amino acids, fatty acids, mineral elements, and vitamins in muscles by biochemical methods. The results showed that the contents of water, crude protein, crude fat, ash, and total sugar in the wet muscles were 76.10%, 19.01%, 1.20%, 1.71%, and 1.98%, respectively. A total of 18 amino acids were detected in muscle, the total amino acids (TAA) accounted for 64.65% of the dry samples. The content of essential amino acid (EAA) reached 22.97%, and the ratio of essential amino acids to total amino acids and total nonessential amino acids (NEAA) was 35.53% and 69.42%, respectively. The amino acid composition was considered suitable. The level of four flavor amino acids was 25.02%, and the flavor was delicious. According to the amino acid score (AAS) and chemical score (CS), the first limiting amino acid was Cystine plus Methionine, and the essential amino acid index (EAAI) was 68.02. The amino acid composition was relatively balanced. The contents of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were 43.02%, 6.59% and 42.39%, respectively. The content of PUFA was relatively high, of which the content of EPA plus DHA reached 32.64%, this has a high health value to the human body. In addition, the muscles of S. lessoniana were rich in mineral elements such as K, Zn, and Se, and vitamins such as VB6 and VB3. In conclusion, S. lessoniana was shown to be a high-quality aquatic product with a comprehensive nutrient composition and a high nutritional value.

Abstract:Bioturbation is important for biogeochemical processes of sediments. The associations between bioturbation and changes in sediment environment have been well studied. However, little information is available regarding the behavior of the infauna, which is a major factor affecting sediments. In the present study, sediments at the low, middle, and high tidal zones were collected at different depths (above 10 cm: sediments 1#, 2#, and 3#; below 60 cm: sediments 4#, 5#, and 6#) from the natural habitat of Perinereis aibuhitensis Grube to study their choice behavior in two devices (I and II). The contents of organic matter, carbon, nitrogen, and hydrogen sulfide in different sediment types were also examined. In device I, there was no difference in organic matter contents across different sediments (P>0.05). Carbon contents in sediments 1# and 4# were significantly higher than those in sediment 2# (P<0.05). The nitrogen content in sediment 4# was significantly higher than those in sediments 2# and 6# (P<0.05). The contents of hydrogen sulfide significantly differed across different sediments (P<0.05). Hydrogen sulfide concentration in the pore water of sediment 1# was significantly higher than those of other sediments (P<0.05). Two-way analysis of variance showed that different P. aibuhitensis sizes and sediment types had no significant effects on the choice behavior of P. aibuhitensis (P>0.05). Size affected the time to search for different sediments (P<0.05), while sediment type showed no effect on search time (P>0.05). In device II, the content of organic matter in sediment 6# was significantly higher than those of sediments 1# and 2# (P<0.05). The contents of carbon and nitrogen in sediment 6# were significantly higher than those in other sediments (P<0.05). The content of hydrogen sulfide in sediment 2# was significantly higher than that in other sediments (P<0.05), and hydrogen sulfide concentrations in pore water significantly differed among different sediments (P<0.05). Two-way analysis of variance showed that different P. aibuhitensis sizes and sediment types significantly affected the movement distance of P. aibuhitensis (P<0.05). Moreover, small P. aibuhitensis were more active and sensitive and preferred sediments with lower hydrogen sulfide content. When P. aibuhitensis drilled inside the sediment, they tended to choose sediments with lower organic matter content. In general, P. aibuhitensis exhibited a slight selectivity for different natural sediment types.

Abstract:Babylonia lutosa is a marine shellfish that has high economic values. In recent decades, the natural resource of B. lutosa has declined due to the environment destruction and overfishing. To further understand the level of genetic diversity and population genetic structure of B. lutosa, we have evaluated the information characteristics of B. lutosa’s microsatellites, after obtaining transcriptome sequences using MISA software. The results show that a total of 16342 microsatellites and 181 nucleotide repeat motifs were identified. Different types of repeat microsatellites had considerably different distribution characteristics. Mononucleotide and dinucleotide microsatellite repeating units were the most abundant in the B. lutosa transcriptome, in which 6 repeats of AC/GT (70.58%) were the dominant repeating dinucleotide units. The length of the dominant repeating units was 12~20 bp (63.95%) and 21~25 bp (9.14%), respectively, and the average length was 18.4 bp. Among the 50 designed primer pairs, 23 proved to be polymorphic microsatellite markers in the B. lutosa wild populations (WP). The results showed that the allele number of these microsatellites ranged from 2 to 7. The expected heterozygosity (He) and observed heterozygosity (Ho) ranged from 0.190 to 0.937, and 0.065 to 0.936, respectively. The polymorphism information content (PIC) ranged from 0.061 to 0.777. The He values of the WP and breeding population (BP) were 0.491 and 0.544, respectively. The Ho values of the WP and BP populations were 0.477 and 0.564, respectively. The PIC values for WP and BP were 0.541 and 0.407, respectively. There were 13 population loci that were heterozygote excesses in WP, and 13 population loci in BP. The genetic differentiation index (Fst) ranged from 0.001 to 0.655, with an average value of 0.053 (0.05

Abstract:Cyanobacterial blooms occur frequently in aquaculture ponds during summer, which has brought great losses to the aquaculture industry. Physical salvage and chemical reagents were previously used to kill the harmful algae and improve the water quality. However, physical salvage is time-consuming, and chemical reagents easily cause more pollution. Thus, there is currently greater consideration to regulate the water quality by using ecological strategies, such as the inhibition of cyanobacteria by submerged macrophytes. Because of its ecological safety, this method is being increasingly applied in eutrophic water bodies. In the present study, coculture simulation experiments were used to observe the effects of submerged plants (Ceratophyllum demersum and Potamogeton pectinatus) on the phytoplankton composition and water quality of ponds (i.e., the concentrations of total nitrogen, total phosphorus, soluble nitrogen, soluble phosphorus, nitrate, and ammonia, and chemical oxygen demand) during cyanobacterial blooms. The results showed that when compared with the control, C. demersum and P. pectinatus could significantly reduce the concentrations of nitrogen and phosphorus in the water column, with significant differences detected between the control and P. pectinatus treatment; inhibit the growth of cyanobacteria, especially of Oscillatoria sp. and Microcystis sp., with the effects of P. pectinatus being greater than those of C. demersum (compared with the values in the control pond, the algal density, biomass, and Chl.a content decreased by 93.6%, 98.9%, and 60.5%, respectively, in the P. pectinatus-treated pond and by 72.5%, 86.8%, and 54.3%, respectively, in the C. demersum-treated pond); and promote the phytoplankton biodiversity of the ponds. At the end of the experimentation, the biodiversity in the ponds treated with C. demersum and P. pectinatus increased by 98.4% and 50.3%, respectively, relative to that in the control pond. The results of this study provide a theoretical basis for the future restoration and remediation of eutrophic waters.

Abstract:In order to understand the current status of drug-resistant bacteria and drug-resistant genes in the sea cucumber, seedlings were collected from 6 aquaculture farms in Shandong Province. The number, proportion and species of antibiotic-resistant bacteria in the intestinal were tested. Meanwhile, the distribution of seven drug-resistant genes of four classes of antibiotics was analyzed by quantitive real-time PCR (qRT-PCR). The results were listed as follows: All of the six kinds of antibiotic-resistant bacteria are detected at all six sampling sites. According to the proportion of drug-resistant bacteria to total cultured bacteria, the top three highest proportions are mequindox, nalidixic acid and tetracycline-resistant bacteria, with the proportions are 0.05%~40.06%, 2.16%~39.94% and 0.06%~23.15%, respectively. The proportion of florfenicol, gentamicin and streptomycin-resistant bacteria range from 0.01% to 4.15%. The 98 antibiotic-resistant bacteria strains are identified as 4 phyla, 5 classes and 30 genera based on 16S rDNA sequence. The top 3 genera are Vibrio, Bacillus and Psychrobacter with the detection rates of 15.30%, 13.27% and 12.25%, respectively. The components of drug-resistant bacteria species varied at different sampling sites, and there are compound antibiotic-resistant strains in Vibrio, Bacillus and Psychrobacter genera. The abundances of seven antibiotic resistance genes in six samples show that the contents of different antibiotic resistance genes against the same antibiotic are significantly different. There is significant correlation between the relative copy number ratio of aminoglycoside resistance gene (aadA) and the proportion of streptomycin and gentamicin-resistant bacteria. The correlation between the abundance of the other six genes and the proportion of drug-resistant bacteria is not significant. The results indicate that there is a certain risk of drug-resistant bacteria and drug-resistant genes in the seedling stage of sea cumber.

Abstract:A bacterial strain with high denitrification activity isolated from a shrimp pond was designated strain 2906. The effects on denitrification efficacy of environmental conditions including carbon/nitrogen (C/N) ratio, pH, and salinity were determined using sucrose and ammonia as carbon and nitrogen sources, respectively. The effects of carbon sources (glucose, sucrose, maltose, sodium succinate, sodium citrate, lactose and soluble starch) on the denitrification efficacy of strain 2906 were assessed. The maximal NH4+-N removal rate of strain 2906 was achieved at a C/N ratio1520, an initial pH of 78, and salinity of 515. Sodium citrate was the optimal carbon source, with which the NH4+-N removal rate reached 100%. In the process of denitrification, there was a strong correlation between bacterial density and denitrification efficiency (R=0.94). A sequencing and phylogenetic analysis of the 16S rRNA of strain 2906 showed it was closely related to Pseudoalteromonas spp. The biosafety of strain 2906 was also tested on juvenile shrimp through an immersion challenge, with the result showing that the 50% lethal concentration of strain 2906 for juvenile shrimp was 2.8×107 CFU/ml, which indicates that strain 2096 has no virulence to shrimp. The results provide a theoretical basis for the development and utilization of the bacterium.

Abstract:Spotted knifejaw (Oplegnathus punctatus) is a new species of mariculture fish with high economic value in China. In August 2017, an outbreak of unknown etiology occurred in spotted knifejaw juveniles cultured in the offshore cages of a marine fish farm. Investigation showed that the water temperature was 26℃~28℃ and that the infected fish were mainly juveniles at the age of 4~5 months. The total length of the diseased fish was (16.3±1.6) cm and their body weight was (156.9±37.0) g. The cumulative mortality of 800000 juveniles was more than 90% within 2 weeks of disease onset. Clinical examination showed that the diseased juveniles had no obvious apparent lesions but had poor vitality and rapid breathing. Necropsy findings included a swollen and brittle spleen, kidney erosion, and hemorrhagic spots in the liver. No parasites were detected, and no pathogenic bacteria were isolated from the liver, spleen, and kidney of the diseased fish. Histopathological sections showed a large number of enlarged cells (about 20 μm in diameter) in the spleen and kidney tissues of diseased fish. In addition, a large number of hexagonal virus particles with a diameter of about 145 nm were observed in the electron microscope sections of the spleen and the body kidney. The spleen homogenate of naturally infected fish was filtered and injected intraperitoneally into healthy fish at a water temperature of 26℃. The fish in the infected group began to die after 10 days post infection (dpi), and the cumulative mortality reached 95% within 14 dpi. Artificially infected fish showed symptoms similar to those in naturally infected fish, and a large number of enlarged cells and similar virus particles could be observed in the spleen and kidney tissue sections. Severe iridovirus infection was detected in the spleen and kidney tissues of naturally and artificially infected fish using specific PCR primers. The major capsid protein gene (MCP) of the iridovirus, at 1362 bp in length was cloned and sequenced, and the phylogenetic tree of the family Iridoviridae was constructed. The results showed that the virus belonged to the red sea bream iridovirus (RSIV) of genus Megalocytivirus in the family Iridoviridae. Based on the results of epidemiological investigation, clinical symptom observation, etiological detection, histopathological observation, artificial infection experiments, molecular biology diagnosis, and viral phylogenetic analysis, it was confirmed that the disease causing the mass death of spotted knifejaw was Megalocytivirus disease, and that the pathogen was a strain of RSIV. This study demonstrates for the first time that RSIV can cause large-scale death in the spotted knifejaw. Furthermore, the research results provide an important reference to diagnose and control viral diseases in the spotted knifejaw.

Abstract:Proteases are a class of enzymes that hydrolyze proteins by cutting protein peptide bonds in vivo, controlling protein size, composition, spatial conformation, and their final degradation. Physiological activities and diseases in organisms are closely related to proteases, such as those for digestion and absorption of food, blood coagulation, hemolysis, anti-inflammatory, blood pressure regulation, cell differentiation, cancer metastasis, and activation of physiologically active peptides. Serralysin-like belongs to the subfamily of zinc metalloprotease M10B and secretes by a variety of pathogenic gram-negative bacteria. It is a key virulence factor for some diseases. Serralysin-like inhibitor can the inhibit target serralysin-like in vitro. The inhibition of bacterial virulence factors presents an antimicrobial strategy that is non-destructive, by attenuating virulence mechanisms without directly challenging bacterial cell viability. The serralysin-like MP and its inhibitor LupI studied in this paper are all secreted by the marine microbial Flavobacterium sp. YS-80-122. We purified the protease MP, inhibitor LupI and MP-LupI complexes. The protease MP was purified by affinity chromatography while the inhibitor LupI was purified by size exclusion chromatography and ion exchange chromatography. After being tested and concentrated, the two were mixed according to the equimolar ratio to obtain the MP-LupI complexes. Finally, purified MP-LupI complexes by size exclusion chromatography to obtain an electrophoresis-purified, single-purification sample of about 95 μl of MP-LupI complexes with a protein content of 20 mg/ml. The MP-LupI complexes were crystallized and successfully obtained under two conditions: 0.1 mol/L DL-malic acid, pH 7.0, 12% (w/v) PEG 3350 to obtain a rhombohedral crystal of MP-LupI complex and 0.2 mol/L NaCl, 0.1 mol/L MES, pH 6.5, 10% (w/v) PEG 4000 to obtain an prism crystal of the MP-LupI complex. The X-ray diffraction data was collected from the Shanghai source to obtain high-quality diffraction data with a resolution of 2 Å. The unit cell space was P1 21 1. The unit cell parameters are a = 51.66 Å, b = 51.85 Å, c = 102.14 Å, α=γ=90° and β=97.68°.

Abstract:Nonspecific immunity plays a vital role in pathogen infection in fish. The bacterium Streptococcus agalactiae is an important pathogen in tilapia cultivation, but its effects on tilapia blood and hepatopancreatic biochemical indices are unclear. We injected three strains of Nile tilapia (Oreochromis niloticus)  a disease-resistant genetically improved farmed tilapia (GIFT) strain, a GIFT Baigui strain, and hybrid tilapia (O. aureus × O. niloticus)  with S. agalactiae, then monitored superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), phosphatase (AKP), lysozyme (LZM), and total antioxidant capacity (T-AOC) enzyme activities in blood and hepatopancreatic tissues. Mortality rates in the hybrid tilapia, GIFT disease-resistant strain, and GIFT Baigui strain were 55.4%, 60.5%, and 78.6%, respectively. Post-infection, enzyme activities of ACP, AKP, SOD, and T-AOC in hepatopancreatic tissues of all strains changed, first increasing then decreasing, and AKP and CAT activities after 24 h of infection were significantly higher in the hybrid tilapia than in the two GIFT strains. ACP, AKP, LZM, CAT and T-AOC enzyme activities in blood increased after infection, but SOD activity decreased; ACP, AKP, CAT and T-AOC activities in the hybrid strain were significantly higher than in the two GIFT strains. Using activity changes of the six enzymes and infection mortality rates, we identified AKP and CAT as potential indicators of streptococcal resistance in strains of tilapia.

Abstract:In order to study the expression profile of mannose-binding lectin (MBL) in response to the stress of heavy metal cadmium (Cd2+) in the ridgetail white prawn, Exopalaemon carinicauda, changes of MBL expression level during 96 h of stress were analyzed in the tissue of hepatopancreas. Five Cd2+ stress concentrations (0, 0.01, 0.0175, 0.021 and 0.0278 mmol/L) were set up in the experiment, and the samples were taken at 8 time points, i.e. 0, 3, 6, 12, 24, 48, 72 and 96 hours after Cd2+ stress. The real-time fluorescence quantitative results showed that under the stress of high concentration of Cd2+ (0.0175, 0.021 and 0.0278 mmol/L), the expression level of MBL gene in the ridgetail white prawn increased first, then decreased after reaching the peak at 72 h. There are significant differences between experimental group and control group at all the stress time points. When the cadmium ion was 0.01 mmol/L, a lower concentration, the expression level of MBL gene in the ridgetail white prawn tends to decrease from the beginning. Further interfering down the expression level of MBL using the RNA technology, it was discovered that the mortality rate of Cd2+ stressed individuals were higher than those of the control group. This study showed that MBL potentially took part in the protection of E. carinicada from Cd2+ stress.

Abstract:Seagrass is a kind of higher angiosperm that originated as a terrestrial plant and over time became adapted to a marine environment. Seagrass beds have important ecological and economical value in that they provide habitats and feeding areas for diverse marine fauna, playing a key role in establishing a flourishing marine ecosystem. From 1993 to 2003, the seagrass acreage lost reached 2.6 × 106 hm2. The first estimated acreages of seagrass beds were recorded in 1879, and based on historical records, it is estimated that more than 5.1 × 106 hm2 of seagrass beds have completely disappeared. With the severe decline of seagrass beds and the public’s recent awareness of their ecological functions, seagrass bed ecological restoration has become one of the more important coastal, environmental engineering projects. Habitat enhancement is the main method utilized in seagrass bed restoration. Currently, seagrass bed restoration is in urgent need of well-organized planning, and large-scale artificial propagations have become vital to current habitat restoration. In order to significantly increase the quantity and efficiency with which seagrass is propagated, this study was to understand the characteristics of seagrass shoot clonal propagation, and determine what techniques would allow efficient plant propagation. In order to achieve highly efficient seagrass shoot propagation, it is necessary to: 1) Promote growth and propagation of key factors; 2) Construct and implement an artificial propagation platform; and 3) Disseminate the growth and propagation planting technique. In this study, the current state of research and knowledge of shoot propagation and planting of seagrasses was reviewed, the environmental factors affecting the growth and development of seagrass shoots was summarized, and the effect of planting space, planting time, and fertilization on the seagrass shoot growth and production was discussed. In addition, the key problems existing at present were summarized. Given the advances in research and public desire to restore damaged ecosystems, there is strong potential for large-scale restoration of damaged seagrass beds along the coast of China in the future, and the summaries provided here will hopefully be a useful reference to these projects.

Abstract:Aquatic products may be contaminated by microorganisms during storage and transportation, leading to product spoilage and quality deterioration. Aquatic products are prone to deterioration by decomposition, putrefaction, degradation of proteins and amino acids, and production of basic amines. Pretreatment by sterilization is required to reduce the effects of microorganisms on the quality and shelf life of aquatic products during processing and storage. The mechanisms, main advantages, and disadvantages of common water treatment methods, including electrolysis or ozonation of water and use of chlorine dioxide solution were compared and analyzed. The research focused on the application of ozonated water in the sterilization and preservation of aquatic products. Ozone exhibits strong oxidizability as well as bacteriostatic and bactericidal effects. In addition, treatment with ozonated water is convenient and plays an important role during storage and preservation by spraying, immersion, and flowing water treatment. Ozonated water is widely used to bleach and decolorize fish fillets and surimi products and to remove strong scents. Additionally, it is also used for product sterilization and preservation, and for cleaning and disinfection of processing equipment. However, ozonated water is associated with poor stability, which may affect its sterilization ability. Therefore, the combination of ozonated water with other preservation technologies may prolong the shelf life of aquatic products to a greater extent than that by ozonated water alone. The combination of ozone water with slurry ice, modified atmosphere packaging, and other freshness preservation methods were described. Researchers should consider using orthogonal experiments or response surface methodology to identify the optimal treatment concentration and duration, and to develop optimal process parameters based on the individual characteristics of different aquatic products.