Abstract:As the first pilot site of the Globally Important Agricultural Heritage System (GIAHS), Qingtian rice-fish co-culture system has attracted more and more attention due to its unique advantages. In order to better conserve the first GIAHS project in Asia, we investigated the effects of different rice planting densities on soil fertility of Qingtian rice-fish co-culture systems. The results showed that the pH value of the tested paddy soil varied between 5.50-6.13, which was weakly acidic. The contents of soil nutrients (such as the soil total nitrogen, soil organic matter, available phosphorus and available potassium) first decreased and reached the lowest values at the booting stage, then recovered to the similar higher levels with the initial before rice harvesting, indicating that the activity of the field fish and perhaps fish feces could help maintain the soil fertility. According to the results of Grey relational analysis (GRA) between rice yield and soil nutrient, it was found that soil pH and available potassium were most closely related to rice yield. Compared with the rich nitrogen, available phosphorus and soil organic matter, the less suficiently available potassium and low pH value might be responsible for the limited growth and yield of rice. Rice planting densities were not found to significantly affect soil fertility in Qingtian rice-fish co-culture system in this growing season.

Abstract:This study was carried out to test the effect of acute ammonia stress on antioxidant enzyme activity and mRNA expression levels of HSP70 and HSP90 genes in tissues of Pelteobagrus fulvidraco, and 360 P. fulvidraco [(17.25 ±0.05) g] were randomly exposed to ammonia: 0 (control), 5.70, 28.50 and 57.00 mg/L total ammonia concentrations for 96 h, respectively. Experimental fish were sampled at 0, 12, 24, 48 and 96 h. The results showed that the superoxide dismutase (SOD) activity in liver of fish in low and medium concentration groups increased from 0 to 6 h, and then gradually decreased, but high concentration group continued to decrease. Malondialdehyde (MDA) content in liver of fish in low, medium and high concentration groups was significantly increased throughout the 96-h period; at hour 3, SOD activity in liver of fish in high concentration group was the lowest, while MDA content was the highest. Liver catalase activity increased significantly at hour 24. In low, medium and high concentration groups, the mRNA expression of HSP70 in liver of fish decreased from 0 to 6 h, and then gradually increased, and that in gill of fish continued to increase, but that in brain of fish continued to decreased. The mRNA expression of HSP70 in liver and brain of fish was significantly lower than that of control group at hour 3, but that in gills is opposite. Compared with HSP70, the mRNA expression of HSP90 in liver and gill of fish in high concentration group reached the highest value at hour 24. The results showed that the antioxidant enzymes activity was inhibited by different concentrations of ammonia, and the reason is related to the accumulation of MDA. Compared with HSP90, the mRNA expression of HSP70 in P. fulvidraco was rapidly up-regulated after the occurrence of ammonia stress, and this physiological regulation mechanism suggests that HSP70 plays a more important role in the response to acute ammonia stress.

Abstract:The genetic structure of species plays an important role in inferring population dynamics such as effective population size, geographical distribution, gene flow and population genetic differentiation. In order to reveal the genetic diversity level and genetic structure of Leiognathus equulus populations, a total of 92 individuals from 3 populations were collected and analyzed by control region fragments. The length of the control region fragment was 393 bp. A total of 32 haplotypes were detected for 92 individuals and 7 haplotypes were shared. The haplotype and nucleotide diversity of Xinzhu population was lower than that of two populations from Hainan. The whole haplotype and nucleotide diversity was 0.61±0.12 to 0.86±0.05 and 0.003 3±0.002 4 to 0.005 3±0.003 4, respectively. Two clades (Clade A and Clade B) were obtained in the neighbor-joining tree and median-networks. Clade A consisted of 22 haplotypes, which all were from Hainan populations; Clade B consisted of 10 haplotypes, which all are from Taiwan except Hap13. The haplotype and nucleotide diversity of Clade A was 0.81±0.04 and 0.003 7±0.002 5, and the haplotype and nucleotide diversity of Clade B was 0.69±0.10 and 0.003 3±0.002 4. Significant genetic differentiation was detected between Xinzhu population and the other two populations. The results of AMOVA showed that most genetic variation occurred among groups, which account for 75.37%. Tajima’s D and Fu’s F_s statistics for two clades were significantly negative for these two clades, which rejected the hypothesis of selective neutrality. The mismatch distribution of L. equulus appeared to be unimodal for two clades, and closely matched the expected distributions under the sudden-expansion model. The expanding time may be nearly 52 500-105 000 and 67 600-135 200 years ago in the late Pleistocene for two clades. The present phylogeographic pattern of L. equulus populations may be the combination of historical factors and current factors. The isolation of the Pleistocene glacial land bridge resulted in genetic differentiation of the populations and the isolated population had a secondary connection. Geographical distance hinders gene exchange and promotes the formation of the existing distribution pattern of L. equulus populations.

Abstract:When breeding for some specific parameters, relevant parameters always undergo covariation. A selective breeding program has been operated on Eriocheir sinensis for different mature time and culture performance equally since 2011, but crabseed quality of selected populations has not been evaluated yet. This study was aimed to evaluate the crabseed quality of two selected populations (early-maturing strain: EM, late-maturing strain: LM) compared to pond-reared crabseed without selection (control). The results showed that: ① The final weight and specific growth rate (SGR) of two selected populations were both relatively higher than those of the control all through the experiment, and the average weight of EM was lower than the LM between August and November, but no significant difference was observed. ② The order of survival rate and final yield was EM > LM > control with significant difference in yield of females, the order of precocious rate was opposite with significant difference in females as well. ③ As for the immune index, the levels of alkaline phosphatase (AKP), acid phosphatase (ACP) and total antioxidant capacity (T-AOC) in hepatopancreas and hemolymph of both selected populations were higher than the control, with significant difference of ALP level in hepatopancreas of females and significant difference of T-AOC level in hemolymph of males. ④ The injection of Aeromonas hydrophilia resulted in both lower (29.50% and 22.94%, respectively) cumulative mortalities of two selected populations. In conclusion, selection for different mature time and culture performance of E. sinensis also presented an effective improvement in their crabseed quality.

Abstract:A plenty of non-coding RNAs (ncRNAs) have been identified through the application of high-throughput analysis of the transcriptome, and this has led to an intensive search for possible biological functions attributable to these transcripts. In this study, the gonad tissue of the two-year-old Crassostrea gigas of same family cultured in Rizhao Huanghai area was used to identify a large number of miRNA, lncRNA and circRNA by small RNA-seq and RNA-seq, and their biological characteristics were analyzed. The results showed that, with Danio rerio as a reference, 25-30 known miRNA matures and 51-63 known miRNA precursors was obtained, 53-71 new miRNA matures and 53-77 new miRNA precursors were predicted. The length of miRNA in C. gigas ranged from 18-26 nt, where the largest number was in the 20-22 nt and the first nucleotide position tended to be U. 2 302-2 349 known lncRNA transcripts were obtained, and 20 083-24 114 new lncRNA were predicted. Among them, the percentage of the intergenic lncRNA, intronic lncRNA and antisense lncRNA was 29.0%, 62.1%, and 8.9%, respectively. The data showed that genomic characteristics of lncRNA in C. gigas were similar to those of other eukaryotes. Compared with mRNA, the transcript and open reading frame of lncRNA were much shorter at length and much lower at expression level. 383 circRNA transcripts were obtained, of which the average percentage of 88.54% came from exon, 4.51% came from intronic and 6.95% came from intergenic. The data showed that the endogenous circRNA have a lot of miRNA target sites. This study revealed the basic biological characteristics of miRNA, lncRNA and circRNA in C. gigas. The results laid the foundation for the subsequent research on the expression rules and biological function of regulatory non-coding RNA in C. gigas.

Abstract:Growth traits of mud clam (Tegillarca granosa) are quantitative traits. In order to acquire some reliable molecular genetic markers for growth-related traits, the correlation analysis of 12 SSR markers and growth-related traits in T. granosa were analyzed using 150 samples with the fourth-generation selected line of T. granosa. And the correlation and path analysis method were used to analyze the effect of phenotypic character on body weight. Then results showed that the correlations among the quantitative characters reached extremely significant levels. The shell height showed the greatest correlation coefficients to the body weight (0.930). However, the result of the path coefficient analysis showed that the highest path coefficients were the shell width (0.432). The results of the determinant coefficients were consistent with the results of the path analysis. In addition, a total of 61 alleles for the 12 loci were detected (3-8 alleles for each locus), and the average number of alleles and effective number of alleles were 5.083 and 3.038, respectively. The average observed heterozygosity (H_o), expected heterozygosity (H_e) and polymorphism information content (PIC) was 0.528, 0.646, and 0.594, respectively, both of which indicated that the population genetic diversity was high. Correlation analysis between genotypes of SSR and growth traits indicated that 3012-2 had a significant impact on shell length, shell width and shell umbo width. Locus 3564 showed a significant influence on shell height and radial rib width. Locus 2692 showed a significant correlation with shell length. Our results could provide theoretical basis for the study of genetic improvement and selective breeding of T. granosa.

Abstract:The clam Cyclina sinensis is an economically important shellfish in China, and it is urgent to explore the excellent germplasm resources of C. sinensis. This requires the support of accurate genetic and biological basic materials. In order to accurately understand the number of chromosome and its karyotype composition of C. sinensis, we prepared diploid chromosomes and haploid chromosomes from the gill tissue and mature gonad of C. sinensis, respectively. The results showed that the chromosome number of diploid was 2n = 38 and haploid was n = 19. On the basis of the above experiments, the well dispersed visual field of diploid chromosomes was measured by microphotography, and the karyotype was analyzed. The result of experiment indicates that the karyotype is 11m+6sm+2st, NF=76, and no heterosome and satellite chromosome were found. In this study, the haploid chromosome was successfully prepared by the mature gonads of the calm, which was to provide a new idea for molluscs chromosome studying. Moreover, the new discovery on chromosome number and karyotype of C. sinensis makes up for the results of previous studies, which was 2n = 36. And it contributed to the more correct direction of phylogenetic research and genetic research of bivalve molluscs, also provided more scientific basis for whole genome sequencing assembly of C. sinensis.

Abstract:The purpose of the experiment was to explore the effects of different riboflavin levels on the growth performance, body composition, and immune parameters of Schizothorax prenanti. Six purified diets were formulated to contain 0.67、2.96、5.83、9.05、11.58 and 23.42 mg/kg riboflavin, respectively. S. prenanti with an average initial body weight of (10.54±0.01) g were fed the diets for 8 weeks and each diet had 3 replicates with 30 fish per replicate．The results showed as follows: the weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of the fish increased first and then decreased with the increase of riboflavin level, and reached the maximum and significantly higher in the 5.83 mg/kg group than those of the other groups; the feed coefficient ratio (FCR) decreased first and then increased, the lowest in the 5.83 mg/kg group and significantly lower than the other groups; riboflavin significantly affected the survival rate of the test fish (SR), liver index (HSI) and visceral index (VSI); riboflavin significantly affected the crude fat content of whole fish, but had no significant effect on crude protein, moisture and ash content. The amount of riboflavin in the liver increased with the increase of the riboflavin level; the liver D-α-amino oxidase (DAAO) activity reached the highest in the 5.83 mg/kg group. The activities of serum lysozyme (LYZ) and catalase (CAT) increased first and then stabilized with the increase of riboflavin. The content of malondialdehyde (MDA) decreased first and then stabilized with the increase of riboflavin level. It indicated that riboflavin can effectively improve the digestibility of feed, improve the immunity and antioxidant capacity, and promote its growth. Considering the best growth performance and immune ability of S. prenanti, 5.32-5.89 mg/kg of riboflavin would be the suitable level.

Abstract:In this study, the full-length cDNA and DNA of a γ-type carbonic anhydrase (CA) gene was obtained from the gametophyte of Saccharina japonica by the rapid amplification of cDNA ends (RACE) technique. The results showed that the cDNA of this gene was 1 618 bp in length, encoding a protein consisting of 305 amino acids, and the genomic DNA was 11 812 bp long, with 6 introns, so that the open reading frame (ORF) was divided into 7 exons. It had a gamma-CA domain and a unique LβH domain. The Neighbor-joining and the Maximum Likelihood phylogenetic tree constructed from the deduced amino acid sequences of 36 CAs showed that the cloned CA was clustered with other γ-CAs. Therefore, the gene was designated Sjγ-CA. In order to understand the function of the encoded protein, the ORF of Sjγ-CA was subcloned and ligated into the expression vector pET-28a to generate pET28a-SiγCA. Subsequently, this construct was introduced into Escherichia coli BL21 for the heterologous expression of target protein. The recombinant Sjγ-CA was purified by affinity chromatography. After SDS-PAGE electrophoresis, Western blotting analysis and mass spectrometry, the purified recombinant Sjγ-CA was identified. The activity of rSjγ-CA in the hydration reaction of CO₂ and HCO₃^- was detected by electrode method and its specific activity was 0.82 U/mg. The hydrolysis of p-nitrophenyl acetate was detected by spectrophotometer, but no esterase activity was detected. Sjγ-CA was thus identified functionally. This study provides a basis for the subcellular localization of Sjγ-CA in gametophyte and sporophyte cells or tissues of S. japonica.

Abstract:To clarify the physiological effects of intestinal aerial respiration on Paramisgurnus dabryanus, the histopathology of the main respiratory organs of P. dabryanus was investigated by inhibiting the intestinal aerial respiration in this study. The results showed that the inhibition of intestinal aerial respiration could cause the death of P. dabryanus around one week post inhibition. When the fish from the experimental group were moribund, the gills, dorsal skin, anterior intestine, mid intestine, posterior intestine, and rectum were collected and subjected to Hematoxylin-eosin (H.E) staining, Alcian-Blue Periodic Acid-Schiff (AB-PAS) staining and scanning electron microscopy (SEM). The results showed that: ① by H.E staining, the gill terminal of the fish from the experimental group was congested, the blood capillary in the epidermis of dorsal skin were contracted and decreased, and dermis cells were deformed. The plicamucosa of anterior intestine was enlarged; meanwhile the connective tissues were denser in the rectum as well as with the exudation of red blood cells (RBc) in the serosa of posterior intestine. ② by AB-PAS staining, the gills, dorsal skins, posterior intestine, and rectum of the fish from the experimental group revealed an increase in acidophilic vacuoles. Besides, the amount of acid mucoprotein on the lamina propria of the anterior and middle intestines increased in the experimental group. By contrast, the neutrophil mucoprotein in submucosa layer decreased in the experimental group. ③ by SEM, the surface of the gill lamellae was damaged and detached, resulting in wrinkling, together with the increased number of secretory pores on the surface of dorsal skin. More bumps were observed on the inner surface of middle intestine of fish from the experimental group. In addition, the granules on the inner surface of the posterior intestine and the rectum of the fish from the experimental group were increased. Taken together, inhibition of intestinal aerial respiration could cause an increase of the mucous cells and exudation of RBcs in the main respiratory organs, and eventually it could lead to death. In summary, intestinal aerial respiration is the necessary physiological activity of P. dabryanus. The current results will shed a new light on the seeding cultivation and sustainable culture of P. dabryanus.

Abstract:In order to investigate the variations of gene expression of salivary gland in Hirudo nipponia under starvation stress, two libraries of starvation treatment group (D30) and control group (D0) were constructed and sequenced using the Illumina HiSeq 2500 platform (paired-end). After stringent quality control of raw data, 145 981 unigenes were obtained using de novo assembly. The average length of unigenes was 675 bp and N50 length was 1 127 bp. And then all of unigenes were annotated via sequence alignment analysis in CDD, KOG, COG, NR, NT, PFAM, Swissprot, TrEMBL, GO and KEGG databases. The amount of gene expression was also estimated according to TPM (transcripts per million). By comparing the transcriptome data of D30 sample and D0 sample, the differentially expressed genes (DEGs) were screened out with a threshold criteria Q value<0.05 and |Fold Change| > 2. Totally 2 650 DEGs were identified under starvation stress, among which 667 genes were up-regulated and 1 983 genes were down-regulated. Four functional DEGs were selected for qRT-PCR analysis, and the results confirmed that the transcriptome analysis was reliable. In addition, 175 pathways were significantly enriched when all the DEGs were subject to GO enrichment analysis. The results showed that most of the DEGs were involved in cytoplasmic ribosome pathway and ribosome pathway was second. The DEGs were almost down-regulated in these pathways. It was also proved in KEGG enrichment analysis that most DEGs were involved in ribosome pathway was second. The DEGs down-regulated in ribosome pathway can indicate that it reduces protein metabolism under starvation stress. Futhermore, 4 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. The results above mentioned provide important reference information for further study on the molecular mechanism of starvation tolerance of salivary gland and the discovery of pharmaceutical value genes in H. nipponia, and also provide reference for other medicinal leeches.

Abstract:Hepatopancreatic necrosis disease (HPND) of Eriocheir sinensis is a multi-symptomatic disease, characterized by white color and atrophy of hepatopancreas. This study aims to evaluate the main symptoms of HPND, hoping for provide a reference for researchers and clinicians to further unite the diagnosis. In this study we collected 11 individual indicators of E. sinensis in health or suspected HPND in criteria Xinghua and Yancheng Cities, Jiangsu Province. Systematic cluster analysis, principal component regression, and multiple correspondence analysis were conducted in this study. Our study dertermined calculated five leading indicators of HPND, including vitality, shell hardness, muscle fullness, hepatopancreas color, and hepatopancreas index. Further, the symptoms were evaluated as poor vitality, lower shell hardness, atrophy and effusion of pereiopoda, the pale yellow of hepatopancreas color, and the low hepatopancreas index (2%-9%). Besides, the analysis showed that the reduction of body weight, and the darkness of hepatopancreas color and gill color might not be related to the typical symptoms of HPND. The results showed five indicators, i.e. vitality, shell hardness, muscle fullness, hepatopancreas color and index, might be the leading indicators in the disease assessment of HPND.

Abstract:Statolith is one of the most important tissues of the Cephalopods which was usually used in the study of age and growth, population structure and life history and so on. Based on the 513 samples of Sthenoeuthis oualaniensis collected by Chinese falling-net fishery from January to March of 2016 and 2017 in the Xisha Islands waters of the South China Sea, the microstructure and growth characteristics of the statolith were studied. The result of principal component analysis of nine morphologic indices indicated that the total statolith length (TSL), maximum width (MW), lateral dome length (LDL) and wing length (WL) could be used to describe the length growth features of statolith of S. oualaniensis. The analysis of variance (AVOVA) indicated that there was no significant difference in the relationship between the morphologic length and the age as well as the mantle length (ML) between sexes, the relationships between all of the morphologic length (TSL, MW, LDL and WL) and age were best described by the power functions, and logarithm functions were best to describe the relationship between morphologic length and ML by the Akaike’s information criterion (AIC). Both the absolute growth rate (AGR) and instantaneous relative growth rate (IRGR) of all morphologic length except WL tended to be high at young stages, and then decreased with the age increasing until the age from 180 to 210 d, and then decreased after the age of 210 d. It seemed that the age 180-210 d was the growth inflection point of the statolith of S. oualaniensis.

Abstract:Since the outbreak of white spot syndrome virus (WSSV) in prawn in 1990s, WSSV has attracted extensive attention and research due to its wide outbreak range and high mortality. VP19 and VP28 are main envelope proteins of WSSV, which play important roles in the early stage of the prawn WSSV infection. These studies focused on using envelope proteins VP28 and VP19 to prepare submit vaccines to protect the host, which currently creates significant protective effect. This paper reviews the protein structure of VP28 and VP19 and their role in WSSV invasion, and the immune application of VP19 and VP28 vaccines in WSSV invasion, including protein subunit vaccine, DNA vaccine, RNA vaccine, and corresponding antibody. The protective efficiency of different types of vaccines is summarized, and the protective rate of VP19 + VP28 dual-potency vaccine is relatively high, which provides a reference for formulating effective methods of WSSV control.

Abstract:Cephalopod beaks, a main feeding structure, is characterized by special formation of pigment. In this study, we analyzed the main chemical composition and carbon-nitrogen stable isotope ratio of whole beaks of three species and different beak parts of Dosidicus gigas with different pigment formation. The results showed that the protein and catechol content in different parts of the beaks were observably different with rostrum > lateral wall > wing, and the C/N ratio is wing > lateral wall > rostrum. The protein and catechol content of three cephalopods were observably different with Sepiella maindroni > D. gigas > Uroteuthis edulis, and the C/N ratio is U. edulis > D. gigas > S. maindroni. There was a significantly positive correlation between chitosan content and the C/N ratio value in different parts of D. gigas’s beak. There was a significantly negative correlation between protein content and C/N value. The C/N of three cephalopods increased with the increasing of chitosan content and the C/N decreased with the increasing of protein content. When the C/N increased, the chitin content was high, the protein content was low and the corresponding pigment became light, while when the C/N decreased, the chitin content was low, the protein content was high and the pigment is dark. Consequently, the degree of pigmentation results from the proportion of protein and chitin, which provides an important basis for investigating specific foraging strategy and individual ontogenetic feeding variation.

Abstract:This study was designed to research the key compounds of jasmonic acid synthesis pathway in several economic red algae. A liquid chromatography-mass spectrometry (LC-MS) was used to establish the detection method of compounds involved in the biosynthetic pathway of jasmonic acid, and the changes of these compounds were detected in Gracilariopsis lemaneiformis and Pyropia haitanensis when they were subjected to mechanical damage. Jasmonic acid (JA), methyl jasmonate (MeJA), 12-oxy-phytodienoic acid (12-OPDA) and 13-hydroperoxylinolenic acid (13-HpOTE) were extracted with 100% methanol, and the four compounds were separated by XBridge^TM C₁₈ chromatographic column with methanol/water as mobile phase. The results showed that the four compounds could be separated well under optimal conditions. The recoveries of the four compounds were 81.23%-90.25%, and the detection limits of the method were between 0.04 and 0.56 ng/mL, indicating that the high sensitivity of this method. These four compounds in G. lemaneiformis, P. haitanensis and Gracilaria asiatica were analyzed. In addition to no JA and 13-HpOTE detected in P. haitanensis, four compounds were detected in the other two species of red algae. The content changes of these compounds in G. lemaneiformis under mechanical damage stress were analyzed, and it was found that when the alga was stressed, the contents of them were accumulated within a short time, and the responses of them were rapid. After P. haitanensis was mechanically damaged, JA production was detected, but the response rates of these compounds were slower than G. lemaneiformis. These results suggest that some red algae may have a similar pathway of jasmonic acid synthesis to that of plants, and also may participate in the stress response to mechanical injury.

Abstract:In order to better analyze the possibility that the floating Sargassum horneri in East China Sea originated from benthic S. horneri in Zhoushan sea area, and provide an ecological reference for effective tracking and monitoring of floating Sargassum, morphological characteristics of vesicle of S. horneri and its relationship to environmental factors were investigated. The results showed that algae began to have vesicle in September, and have a highest growth rate from March to May. The number of vesicles in the main side branches increased rapidly from March to April, and reached a maximum in April. The number, average length, average wet weight and average volume of vesicles in each month outnumbered in top main side branch than those in middle and bottom main side branches, but the average diameter was relatively uniform. The net effective accumulated temperature needed by S. horneri to complete the life history was 4 434.75 ℃·d. Vesicle germinating net effective accumulated temperature in September was 1 853.47 ℃·d, and development completing net effective accumulated temperature in April was 3 611.25 ℃·d. The sea surface temperature change in the study area is mainly affected by the current changes caused by monsoon activity. The warming condition after a low temperature period of sea water may be the factor that induces the rapid growth of S. horneri, and higher sea temperature may limit algae growth.