Abstract:The ichthyoplankton assemblage structure and succession in the Bohai Sea were studied using multivariate statistical analysis of monthly investigation data collected from April 1982 to May 1983 as background. Based on a pooled analysis of the historical data of fish eggs and larvae sampled from the horizontal trawl surveys during oceanographical expedition cruises over the past 30 years combined with the field survey of current fish habitat conditions, this study aimed to investigate the seasonal and interdecadal variations in the species diversity and key species, and the changing collaboration between the dominant species and the important species in the succession process. Compared with the survey results of 1982~1983, the profiles of the species number and abundance index of fish eggs and larvae during the survey season across different survey times present different degrees of decline (except in winter), and display a tendency towards decline at the beginning and an increase later. The species number of the fish eggs was only one half in the 1980s, and its abundance index was only one tenth in the 1980s. The species number and abundance index of fish larvae were only three fourths in the 1980s, however, these have shown an obvious increasing tendency in the recent years during winter. During the investigation period, species substitution in the early life stages of fish was obvious, otherwise, the substitution rate was significantly accelerated in the recent years. The dominant species of fish eggs did not change significantly during the same season in each investigation period, however, variation in the dominant species in fish larvae was more obvious. The dominance of the continental shelf demersal fish communities with high commercial value decreased sharply, although some species disappeared in recent surveys. The biodiversity index of fish eggs and larvae was higher during the sea temperature rising season and lower during the sea temperature cooling season. The index profiles during the survey season across different survey times showed a tendency of decreasing first and then increasing. Temperature adaptation studies on the spawning stock profiles during the survey season across different survey times showed that species number in each temperature adaptation type showed a tendency of decreasing first and then increasing. Even so, the seasonal or annual percentage of each type remained stable. Habitat studies on the spawning stock showed that the species number of the main habitat type also rose after showing a declining trend first, and presented different degrees of decline. The annual percentage of the continental shelf pelagic-neritic fish increased, in conjunction with the continental shelf demersal and benthopelagic fish decrease. Under the multiple sources of exogenous interference factors, irreversible changes have been taking place in every key link of the recruitment process in the early life stages of the fish, with habitat loss or fragmentation in the Bohai Sea during the past 30 years. Stressors such as overfishing and climate change promote multidimensional niche disturbances in the fish community and structural decline in the fishery resources, while the succession and assemblage structure of the fish eggs and larvae are the embodiment of this development.

Abstract:The Penglai 19-3 oil spill in 2011 is considered as one of the most serious oil spill incidents in the Bohai Sea. However, the effect of the oil spill on benthic crustaceans is unclear. In this study, we analyzed the inter-annual variation in the community structure of crustaceans in the Bohai Sea based on the survey data collected by bottom trawls in summer (August) from 2009 to 2015. A total of 33 benthic crustacean species belonging to 2 orders, 21 families, and 29 genera were captured, which included 16 shrimp species, 16 crab species, and 1 stomatopod. The ecologically dominant species changed every year. However, Oratosquilla oratoria was the most dominant species every year from 2009 to 2015. Both the number of crustacean species and the biomass or individual yields declined from 2009 to 2013, and then increased from 2014 to 2015, synchronously. Cluster analysis (CLUSTER) and multidimensional scaling (MDS) analysis indicated that the crustacean community during 2009 to 2015 can be divided into 4 groups. The single factor analysis of similarities (ANOSIM) indicated that the difference in the community structure of crustaceans was significant (P<0.05). The similarity analysis of percentage (SIMPER) indicated that O. oratoria, Palaemon gravieri, and Latreutes anoplonyx contributed maximum to the intergroup similarity. The community alternating and community migration indices indicated that the stability of the crustacean community was the best in 2010 and the worst in 2013. The results indicate that the benthic crustacean community in the Bohai Sea recovered gradually during 2014 to 2015, three to four years after the Penglai 19-3 oil spill incident.

Abstract:The present study used Nibea albiflora as an indicator species to evaluate the effects of climate change on the early life history of marine fishes. Based on the forecast of the Intergovernmental Panel on Climate Change (IPCC) provided by the Representative Concentration Path (RCP), the emission of greenhouse gases would cause a significant increase in the temperature with a considerable decrease in pH of the oceans by 2100. We compared the growth, development, and mortality of N. albiflora larvae cultured at four different temperatures (24.0℃, 26.0℃, 26.6℃, and 28.0℃) and three different pH (7.80, 7.73, and 7.49). The different temperatures and pH were selected based on the forecast of IPCC in 2100 under two scenarios: scenario 1 with reduced emission of greenhouse gases (RCP 2.6) and scenario 2 with high level of greenhouse gas emission (RCP 8.5). The results showed that it takes approximately 25 d for N. albiflora larvae to become juveniles when cultured at 24℃, whereas it takes just 22 d for those cultured at 26℃~28℃. Furthermore, N. albiflora larvae exhibited higher growth rates at higher temperatures. The mortality of N. albiflora larvae did not show a consistent trend with increase in the surrounding temperature, with the highest and lowest values at 26.6℃ and 26.0℃, respectively. In addition, N. albiflora larvae tended to have a higher mortality at a lower pH, with 3.9%, 19.4%, and 21.7% mortality at pH 7.80, 7.73, and 7.49, respectively. On the contrary, ocean acidification did not have significant effects on the growth of N. albiflora larvae. The present study also predicted that the growth rate and mortality would be 0.71 mm/d and 31.1%, respectively, for N. albiflora larvae under RCP 2.6, and the growth rate would increase to 0.76 mm/d with a 23.9% decrease in mortality under RCP 8.5 by 2100.

Abstract:The successive data of environmental factors such as nutrients and heavy metals in the surface water of Jinghai Bay Trachidermus fasciatus Heckel National Aquatic Germ Plasm Resources Conservation Area in the summer of 2014 to 2016 were used to evaluate the ecological environment quality of the survey area by the comprehensive quality index method. The organic pollution index, the potential eutrophication assessment and the principal component analysis were employed to analyze the water trophic status, the organic pollution, and the main pollution factors. The results showed that the comprehensive quality index of seawater in Jinghai Bay was 1.04~1.69. The water quality in this area was light pollution based on the relationship between the comprehensive quality index and environmental classification. The range of organic pollution index was 4.66~9.23 in 2014 to 2016, which was serious organic pollution based on the relationship between the organic pollution index and the organic pollution classification in seawater. According to the nutrition evaluation model, the nutrients levels of the survey area were rich nutrient in 2014 and potential rich nutrient with limited nitrogen in 2015 and 2016. The principal component analysis showed that DIN, As and Zn were the main environmental factors affecting the water quality of this survey area, which represent the pollution status of Jinghai Bay Trachidermus fasciatus Heckel National Aquatic Germ Plasm Resource Conservation Area from 2014 to 2016. Thus, we should pay more attention to the nutrients, organic pollution and heavy metal (As and Zn) in the process of environmental monitoring and project.

Abstract:The albinism in Japanese flounder aquaculture has become a common phenomenon and influenced its large-scale farming and market value. Based on the albinism and normal Japanese flounder transcriptome and miRNA sequencing, tyrosinase related protein 1(tyrp1) and mmu-miR-143-5p_R+2 (mmu-143) were chosen for expression pattern, target gene prediction and verification analysis. Firstly, tyrp1a and tyrp1b were screened and identified by RACE and phylogenetic analysis. Subsequently, RNA hybrid was used for the prediction of the targeting relationship between tyrp1a and miRNA mmu-143, which was further verified by dual luciferase experiment. Finally, the results of the quantitative real time-PCR (qRT-PCR) showed that the expression of tyrp1a gene in normal skin of Japanese flounder was significantly higher than the albinism skin, and the expression of mmu-143 in normal skin was significantly lower than the albinism skin. The present study identified two transcripts of tyrp1a and tyrp1b from Japanese flounder. Dual luciferase experiment and qRT-PCR analysis confirmed that tyrp1a was the target gene of mmu-143, and mmu-143 affected the albino of Japanese flounder by regulating tyrp1a. The results of this study will be helpful to fully understand the molecular mechanism of Japanese flounder albinism.

Abstract:The effects of variable temperature acute change (21℃, 24℃, 28℃, 32℃ and 35℃) and temperature gradient (rising 1℃ every day) on the antioxidant and digestive physiology in hybrid progeny (Mussaurus spot) of Cromileptes altivelis Valenciennes♀× Epinephelus lanceolatus♂ were studied. The results showed that feed intake and digestive enzyme activity in gradual rising temperature group were higher than those in temperature acute change groups. The temperature acute change groups: the feed intake and digestive enzyme activity were gradually increased from 21℃ to 32℃, but decreased in 35℃ group. The serum SOD activities were increased significantly in most temperature mutations groups except 35℃ group at the 7th day. The liver SOD activities had no significant difference before and after treatment in 21℃, 24℃ and 28℃ groups, but decreased in 32℃ group and increased in 35℃ group. The activities of catalase enzyme (CAT) in serum and liver had increasing trends in 21℃, 24℃ and 28℃ groups, but had decreasing trends in 32℃ and 35℃ groups. The serum MDA content increased at first and then decreased in 32℃ group and the amplitude of variation was significantly larger than other groups with gradually decreased content of MDA. The liver MDA content increased at first and then decreased in all groups. The gradual rising temperature group: The serum SOD and CAT activities increased at first and then decreased. The liver SOD activity had no significant change at the 3rd day but significantly increased at the 7th day, while the liver CAT activity decreased significantly at both the 3rd and 7th day. The MDA content in both liver and serum were gradually declined. The temperature acute change groups: the intestinal tract pepsin and lipase activities were similar in 21℃ and 24℃ groups, which decreased at first and then increased. The pepsin activity gradually increased in other groups, while the lipase activity increased at first and then decreased. The amylase activity maintained at a lower level without obvious change. The activity of pepsin and lipase increased significantly in the gradual rising temperature group, while there was no significant change in the amylase activity. In summary, temperature gradual rising can promote the feed intake and antioxidant ability of juvenile Mussaurus spot, while the temperature acute change can make significant effect on antioxidant system and may cause liver damage overtime. So temperature acute change should be limited to reduce the adverse stress response during the feeding time, and temperature gradual rising can be used to achieve the required conditions.

Abstract:In this research, the chromosomal preparation and karyotype of Epinephelus moara♀ × Epinephelus lanceolatus♂ hybrid progeny (commonly known as Yunlong grouper) were studied. The juvenile fish weighing 60~100 g were injected subcutaneously with 6 μg/g phytohemagglutinin (PHA) and 0.1‰ colchicine. After 21 h, the head kidney and gill were dissected for cell suspensions, slices of their chromosome split phase were made by cold and hot drop method combing with air-drying. After being stained with 10% Giemsa, a clear and complete split phase was screened under a microscope. Chromosomes in 75 mitotic phases were selected and the karyotype analysis was performed. The results showed that the karyotype formula was determined to be 2n=48, 4sm+6st+38t, NF=58 in Yunlong grouper. Hybrids Yunlong grouper had the same chromosome number with their parents (2n=48), but their karyotype was different, indicating the complex chromosomal recombination and mutation occurred in the genetic process. This study provided an important scientific basis for the germplasm identification genetic relationship analysis and crossbreeding of Yunlong grouper.

Abstract:To assess the correlation between the main morphological traits and body weight of Siniperca chuatsi as well as the morphological differences between males and females, 2837 S. chuatsi individuals from the same farm pond were used to measure seven growth-related morphological traits including total length, body height, head length, eye diameter, caudal peduncle length, caudal peduncle depth, and body weight. Correlation analysis, path analysis, and multivariate linear regression analysis were used to analyze the correlation between morphological traits and body weight. A total of 150 males and 150 females were randomly selected to establish multiple regression equations, and 120 individuals were used for gender verification. The results showed the correlation coefficients of body weight to total length and body height are the highest among all the morphological traits. The established regression equations and partial regression coefficient tests indicated no significant correlation of head length or caudal peduncle depth to body weight, which were excluded for further analysis. Path analysis revealed the largest direct effect of total length and body height upon body weight, which was greater than their indirect effect. The highest determinant coefficients of single and pairwise traits to body weight were total length and body height in both single trait determination and pairwise determination. The determination coefficient (∑d) of the four traits was 0.896, suggesting a greater correlation for the four traits to body weight and a higher reference value for the fitted multiple regression equation. A regression equation for the gender was established. Verification results indicated that higher verification accuracy of gender was obtained for individuals with body weight less than 200 g. This study provides an essential reference for determining the evaluation indices for the morphological growth traits of S. chuatsi, and would facilitate the breeding selection.

Abstract:In order to investigate the optimal dietary protein and energy levels of a new juvenile hybrid grouper (Epinephelus lanceolatus♂×E. moara♀), nine diets were formulated containing three dietary protein levels (46%, 50% and 54% crude protein) and three dietary energy levels (20.00 kJ/g, 20.50 kJ/g and 21.00 kJ/g gross energy), the dietary protein-energy ratio was in the range of 22.04 to 26.87 mg/kJ. Juvenile grouper averaging (46.23±0.51) g were fed the test diets for 56 days. The results indicated that the interaction between protein and energy had a significant effect on the weight gain (WG) of grouper (P<0.05), the weight gain rate (WGR) and specific growth rate (SGR) increased with increasing of dietary protein and energy levels (P<0.05). Protein deposition rate (PDR) and hepatosomatic index (HSI) were significantly affected by the interaction between protein and energy (P<0.05). The energy retention rate (ERR) and condition factor (CF) increased with increasing of dietary protein levels (P<0.05), while the protein efficiency ratio (PER) increased first and decreased afterwards (P<0.05). The PER and ERR increased with increasing of dietary energy levels (P<0.05). PDR and PER of D6 group were significantly higher than other groups (P<0.05). The interaction of protein and energy had no significant effects on moisture, crude protein and crude ash of whole fish and muscle (P>0.05). The crude protein of whole fish and muscle increased with increasing of dietary protein levels (P>0.05). Moisture decreased with increasing of dietary energy levels (P<0.05). Crude fat of whole fish increased with decreasing of dietary protein-energy ratio (P<0.05). The interaction of protein and energy had significant effect on the activities of trypsin, lipase and amylase (P<0.05). The pepsin activity increased with increasing of dietary protein levels (P<0.05), and dietary energy levels had no significant effect on it (P>0.05). The trypsin activity of D6 group was significantly higher than other groups (P<0.05), which increased first and then decreased with decreasing of dietary protein-energy ratio (P<0.05). The lipase activity of fish fed diets containing 21.00 kJ/g energy was significantly higher than other groups (P<0.05). According to these results, a diet containing 50% protein and 21.00 kJ/g energy was recommended for efficient growth of juvenile hybrid grouper.

Abstract:This study investigated effects of exogenous Vitamin C (VC) on the membrane time, hatching ratio, survival rate, growth characteristics, digestive enzyme activities and Lipases enzyme activities of spotted halibut (Verasper variegatus) during early development using mature eggs by the method of artificial fertilization. The eggs were divided into five groups with five different VC concentration of 0, 20, 25, 30, and 35 mg/L, respectively. Samples were collected at mature eggs, fertilized eggs, mid-gastrula, crystal appear, mixed nutrition and exogenous nutrition stages. The results showed that 25 mg/L VC significantly improved the hatching ratio, survival rate and growth characteristics and shorten the membrane time, and that VC contributed to the deposition of protein in spotted halibut during early development, and significantly enhanced the activity of pepsin after exogenous nutrition stage (P<0.05). After mixed nutrition stage, VC significantly increased the activity of amylase (P<0.05). After endogenous nutrition stage, VC significantly induced the activity of lipase and trypsin (P<0.05). VC at 25 mg/L increased the activities of lipoprotein lipase and hepatic lipase enzyme during embryo development (P<0.05). The findings indicated that lipoprotein lipase and hepatic lipase genes in zygotes may play functions at gastrula stage when lipid hydrolytic enzymes express to release energy for embryonic development. The results demonstrate that lipase and trypsin priority digest carbohydrates in yolk of mature eggs from spotted halibut to provide energy for embryonic development right after fertilization. The study revealed that appropriate level of VC could improve lipid metabolism during embryo development of spotted halibut.

Abstract:Studies were conducted for further understanding the level of genetic diversity and population genetic structure of Fenneropenaeus chinensis under artificial breeding conditions. Fifteen fluorescence labeled microsatellite primers were used to analyze the genetic diversity and genetic differentiation in wild population (WP) and breeding population (BP) of F. chinensis. The wild population was collected from the western coast of Korean Peninsula (34°30¢N, 127°30¢E), and the breeding population was obtained after selection for successive ten generations. In each population, 40 samples were used for DNA extraction according to the protocol provided by the manufacturer. PCR was performed in a 25 µl reaction and the PCR products were sequenced by Sangon Biotech(Shanghai) Co., Led. Allele data was analyzed by Cervus 3.0.7 and GenALEx 6.502. The results showed that a total of 462 alleles were identified at 15 microsatellite loci, the numbers of alleles (Na) and effective alleles (Ne) were 3~44 and 2~29 in WP and BP, respectively. The polymorphism information content (PIC) ranged from 0.518 to 0.964, and the observed heterozygosity (Ho) values of WP and BP were 0.518 and 0.964, respectively. P-values were calculated to confirm whether the 15 loci deviated from the Hardy-Weinberg equilibrium or not. Of the 30 population loci, there were 11 population loci was heterozygote excess. The Shannon genetic diversity index (H) in WP and BP were 2.786 and 2.399, respectively. The Nei’s unbiased genetic Distance (uD), as well as unbiased genetic Identity (uI) was 0.17 and 0.838, respectively. The Gene Flow (Nm) and Fst value of the two populations were 17.997 and 0.017 (P=0.001), respectively, indicating that there was a low genetic differentiation. Partitioning of the genetic variation revealed that only 7.50% of the genetic variation was among the populations, and the other genetic variation was within the populations. This study showed that the ‘Huanghai No.2’ still has high genetic diversity after selection for successive ten generations, and it also has potential value for further breeding materials

Abstract:Fructose-1,6-bisphosphate aldolase (FBA) is a key glycolytic enzyme involved in both the glycolytic and gluconeogenic pathways. In the present study, fructose-1,6-bisphosphate aldolase from Chinese shrimp Fenneropenaeus chinensis (FcFBA) was cloned and sequenced. The full-length complementary DNA (cDNA) sequence of FcFBA is 2496 bp long, which contains a 79-bp 5¢-untranslated region (UTR), 1319-bp 3¢-UTR, and 1098-bp open reading frame, encoding a polypeptide of 365 amino acids with a molecular mass of 39.8 kDa and a theoretical isoelectric point of 6.6. Multiple sequence alignments showed the high similarity of FcFBA with the aldolases of other arthropods. Phylogenetic analysis showed that FcFBA of shrimp belonged to the same class as that of other arthropod aldolases. The transcript of FcFBA showed the highest expression in the muscle and lowest expression in the hepatopancreas, which indicates that FcFBA is similar to the muscle type (type A) aldolase that acts in the glycolytic pathway. The expression profile of FcFBA in the hepatopancreas, gills, and muscle was modulated when the shrimp were stimulated by white spot syndrome virus (WSSV), which resulted in differential expression of FcFBA. Further, RNA interference (RNAi) was used to analyze the role of FcFBA. After RNAi, the mortality of shrimp after WSSV infection was altered compared with that after phosphate-buffered saline injection. Within 24 h of RNAi, the expression level of FcFBA was significantly down regulated. These results show that FcFBA is inducible and might be involved in the immune response of shrimp.

Abstract:The present study evaluated the effects of white spot syndrome virus (WSSV) infection in Litopenaeus vannamei ‘Renhai No.1’ and Fenneropenaeus chinensis ‘Huanghai No.2’ at various temperatures (24℃, 28℃, and 32℃) using quantitative feeding method (L represents L. vannamei, and F represents F. chinensis). The results showed that the average survival time of group L-24℃, F-24℃, L-28℃, and F-28℃ was (184.05±69.56) h, (101.68±38.45) h, (100.25±26.79) h, and (73.38±22.22) h, respectively. The survival time between two kinds of shrimps at same temperature was significantly different (P<0.05). At the 15th d, the survival rate of group L-32℃ and F-32℃ was 45.74% and 23.47%, respectively. The time of 50% mortality rate of two shrimps at three temperatures was 178 h and 98 h, 98 h and 74 h, 292 h and 78 h, respectively. The time of death peak of two shrimps among three temperatures was 5 d and 4 d, 5 d and 4 d, 10 d and 4 d, respectively. The virus loads in muscle of L-24℃ and F-24℃ at 144 h was (2.97×106±7.44×106) and (8.08×106±3.22×106) copies/ng DNA with the highly significant difference (P<0.01). The virus loads in muscle of L-28℃ and F-28℃ at 144 h was (6.73×106±1.49×106) and (1.20×107±6.15×105) copies/ng DNA with highly significant difference (P<0.01). The virus loads of muscle of L-32℃ and F-32℃ at the 15th d was (5.18×103±4.32×103) and (3.78×104±8.97×103) copies/ng DNA with a significant difference (P<0.05). These results indicates that L. vannamei are more tolerant to WSSV infection than F. chinensis, and the ability of multiplication of WSSV in muscle at different temperatures was 28℃>24℃>32℃

Abstract:In recent years, the fast development of aquaculture has been followed with the increased diseases such as the white spot disease to cause a great loss for the global aquaculture industry. To understand the variations in WSSV ORF14/15 and ORF23/24 and the VNTR in different regions of China in 2015, we investigated 57 samples WSSV-positive on PCR, collected in a disease outbreak area between April and October 2015. The tested samples were from Shandong, Guangdong, Hainan, Zhejiang, Tianjin and Jiangsu. Using specific primers, WSSV-positive samples were genotyped and the amplified fragments were ligated to a T-vector and transformed in Top10 cells. The positive clones were selected and sequenced. After sequencing, the fragments deleted from ORF14/15 and ORF23/24 in different samples were compared with the sequences of Th-96-Ⅱ and TW respectively, and three WSSV VNTR regions in ORF75, ORF94, and ORF125 from different samples were analyzed with the DNAMAN software. The results showed that 35 samples had the products of ORF14/15 amplification, and 14 sample had the products of ORF23/24 amplification. The lengths of the amplified fragments of ORF14/15 were 1270 bp, 1892 bp and 2075 bp, corresponding to deletions of 6530 bp, 5908 bp and 5725 bp, respectively, compared with Th-96-Ⅱ. One type of ORF23/24 was confirmed, and the amplified fragment was 1140 bp, corresponding to a deletion of 12070 bp compared with the TW isolate. Ten fragments were amplified from ORF75, including 1, 2, 3 repeat units with 45 bp, and 1 with 102 bp. Four different ORF94 VNTRs, including 4, 5, 10, or 12 repeat units, were identified. The ORF125 VNTR including 3, 5 or 6 repeat units was identified. The results showed that WSSV exhibited a significant difference in some open reading frames, but a significant stability in others.

Abstract:Clathrin heavy chain protein (CHC), a conserved protein only found in eukaryotes, is one of the essential components of clathrin. White spot syndrome virus (WSSV) infection is one of the main diseases in aquaculture. It belongs to the Whispovirus genus of the Nimaviridae family. WSSV, a rod-shaped, non-occluded baculovirus with large doubled-stranded DNA, can infect shrimps such as penaeid shrimp and crayfish. WSSV has a wide range of hosts in crustacean with high infection and mortality rate, and it causes up to 100% mortality within 3~7 days, which causes great economic loss in aquaculture industry. Recent research showed that the clathrin mediated endocytosis path regulates WSSV infection in the Cherax quadricarinatus hematopoietic cells. However, whether the clathrin heavy chain protein plays an important way in the clathrin mediated endocytosis pathway is unclear. To address this, two primers were designed to clone the LvCHC1 and LvCHC2 according to the two structural domains: clathrin propel repeat (LvCHC1) and clathrin heavy chain repeat homology (LvCHC2) of Litopenaeus vannamei CHC. LvCHC1 and LvCHC2 were cloned into prokaryotic expression vector pBAD/gⅢA and transformed into E. coli TOP 10. The recombinant LvCHC1 and LvCHC2 were successfully obtained by inducing with L-arabinose. The pure LvCHC1 and LvCHC2 were acquired using Co2+ affinity chromatography purification. Mass spectrometry analysis showed the correctness of the recombinant LvCHC1 and LvCHC2. Far-Western blot results indicated that LvCHC1 and LvCHC2 interacted with VP26 and VP37 with higher banding activity with VP26, and that LvCHC1 and LvCHC2 did not bind to VP28N. Taken together, the results indicated that clathrin heavy chain-mediated endocytosis is required for WSSV infection.

Abstract:Portunus trituberculatus, an economically important food species in China, lacks adaptive immunity and can induce effective immune responses to resist pathogen via innate immunity. Toll-like receptors as pattern recognition receptors (PRRs) play crucial role in innate immune responses. The current study found a novel Toll gene named as PtToll4 from P. trituberculatus by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of PtToll4 was 4276 bp, which contains an open reading frame (ORF) of 2685 bp, a 3¢ untranslated region (UTR) of 1252 bp, and a 5¢ UTR of 339 bp. The ORF encoded a polypeptide of 895 amino acids with a predicted molecular weight of 102.5 kDa. PtToll4 is a transmembrane protein that contains several common structural domains of TLRs, such as LRR, LRRCT motif in the extracellular domain and a TIR domain in the intracellular region. The BLAST analysis showed that PtToll4 shared the highest homology with Eriocheir sinensis Toll2. PtToll4 was widely expressed in hemocyte, heart, gill, hepatopancreas, stomach, intestine, muscle, cuticle, and eyestalk, and mainly distributed in the hemocyte and cuticle. A weak expression level was detected in the hepatopancreas. WSSV inflection significantly induced PtToll4 expression in hemocyte at 6 h, and V. parahemolyticus inflection had a little effect on it only at 48 h. Low salinity reduced PtToll4 expression. Our results suggested that PtToll4 may play an important role in innate immune of P. trituberculatus and would provide useful information for the research of immunity regulation in crustaceans.

Abstract:The wild population of Portunus trituberculatus, an important economical species in China, has been dramatically decreased in past decades due to over capturing. To recover wild germplasm resources of P. trituberculatus, the activities of stock enhancement were performed, and the released individuals were about six millions each year in Haizhou Bay of Jiangsu Province. To evaluate the effects of stock enhancement, we utilized molecular markers (mtDNA SNPs) to track the released pedigrees of P. trituberculatus. Previous studies identified 22 SNPs of mtDNA from P. trituberculatus, which were used to trace four released families (on behalf of 0.8~1.2 million released individuals) (A, B, C and D) by HRM (High resolution melt) technology. Nine of 22 SNPs were usable, which resolution melt curve were coincident between female parentages and their offspring. Furthermore, the nine SNPs could be used to distinguish the four families, in which the parentage C, B, A and D could be differentiated by 5, 2, 1 and 1 SNPs, respectively. Our results indicate that the mtDNA SNPs, combined HRM technology, is a wonderful technology to evaluate the effects of stock enhancement of P. trituberculatus.

Abstract:Common cuttlefish (Sepia officinalis) is one of the most important economic and ecological species in the West African coastal waters. Studying the morphology and growth characteristics of its beak is beneficial for common cuttlefish´s development and utilization. This study analyzed beak morphological parameters and their ratios based on the samples collected by the Chinese trawlers in 2015 in coastal waters of West African Mauritania, and studied the beak growth variations in different mantle length groups and different gonadal maturity stages. Morphological parameters of beaks were used to estimate the size of the cuttlefish. The results showed the length ratio indices of the lower beak were not stable, and that the beak morphological parameters of female were significantly higher than the male after eliminating the effect of the sample size differences by dividing the mantle length (P<0.01). The ANOVA and the Least Significant Difference (LSD) analysis showed that morphological parameters of the common cuttlefish beaks significantly increased with the growth of mantle length (P<0.01) and gonad maturity stage Ⅱ~Ⅳ (P<0.01). However, beaks in the gonad maturity stage V were significantly smaller than those in the previous periods (Ⅱ~Ⅳ, P<0.05). Principal component analysis showed that upper hood length (UHL) and lower crest length (LCL) had the highest loading coefficient in the first principal component, and that lower rostrum length (LRL) and lower rostrum width (LRW) had the highest loading coefficient in the second principal component, which can be used for the mantle length estimation. This study provides basic biology information for the classification and ecological research about common cuttlefish in the coast of West Africa.

Abstract:To investigate the influences of different drying methods on the levels of fucoxanthin, a new RP-HPLC method was established to determine the fucoxanthin content in the fresh or dried kelp and sargassum samples. The samples were extracted by methanol and then treated by C18 column solid-phase extraction to remove the other liposoluble components. The separation column was ZORBAX SB-C18(4.6×150 mm, 5 μm), which was held at 35℃. The mobile phase was composed of the gradient changes of methanol and ultrapure water at a flow rate of 0.7 ml/min. The sample loading volume was 10 μl. UV detection was performed at 450 nm. The method showed a good linear relationship between peak area and concentration over the range from 0.11 to 50 mg/L (R2=0.9999). The recoveries of fucoxanthin in three replicates ranged from 92.66% to 109.06%. The limit of detection was 0.03 mg/L. The RSD of precision was 3.46% to 4.61%. This method is simple, high sensitive and accurate. The levels of fucoxanthin in the fresh kelp and sargassum samples were 559.2 mg/kg (dry weight) and 680.4 mg/kg (dry weight), respectively. The fucoxanthin levels of the dried brown seaweed samples were significantly lower than those of fresh samples. All drying methods destroyed fucoxanthin in the seaweed samples, while freeze drying method was found superior to the low-temperature drying (45℃) method and natural drying method. Therefore, the fresh and freeze-drying seaweeds should be ensured to achieve high yield during the large-scale preparation of fucoxanthin. This research would provide the theoretical guidance and technical support for the development and utilization of seaweed fucoxanthin resource in the future.

Abstract:High-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD) was widely used to analyze monosaccharide in botanical and fungal polysaccharides, but lightly used in marine animal polysaccharides. A method has been developed for determining monosaccharide composition and content in sea cucumber polysaccharide by HPAEC-PAD. Purifying sea cucumber polysaccharide were optimized by comparing three deproteinization methods. Optimum chromatographic conditions for the separation of monosaccharide were optimized by comparing eluent concentration on the separation of monosaccharide. The results indicated that the reagent of Sevag removed micro-molecule polypeptide more effectively than others from sea cucumber polysaccharides. The concentration of eluent had significant effect on separating monosaccharide. Eight monosaccharide, such as Fuc, Gal, Ara, GalN, GlcN, Man, Glc, and Lac, were separated using 20 mmol/L NaOH as eluent. GlcUA and GalUA were separated by 160 mmol/L NaOH and 200 mmol/L NaAC as eluent. The linear coefficients of monosaccharide standard were above 0.998. The detection limit of each monosaccharide was between 2.5 and 50 μg/L, and the recovery rates ranged from 83.6% to 113.1%. The method determining neutral sugar, amino sugar and uronic acid in sea cucumber polysaccharides could be used as analysis and quantitative method of monosaccharide in sea cucumber and its products. The composition and content of monosaccharide were significant indices for determining the biological activity of sea cucumber. The established method could provide basic monosaccharide information and promote the polysaccharides research progress in sea cucumber. It could provide references for the establishment of sea cucumber standards, and expand the application of active substances in medicine and other fields.