The peptides encoding ORF domains were synthesized based on the amino acid sequence of lepa and lepb from Cynoglossus semilaevis. The prokaryotic expression vector pEQ30 was used to construct the recombinant plasmids of lepa/pQE30 and lepb/pQE30, which were transformed into Escherichia coli M15 strains and then induced by IPTG to obtain the recombinant proteins of LepA and LepB containing 6 His at the N-terminus. The obtained LepA and LepB polypeptides expressed in form of inclusion bodies had molecular weight of both 16 ku, and the optimum condition for the highest expression of the target proteins were induction by 0.5 mmol/L IPTG at 37 °C for 4 hours. The concentrations of LepA and LepB recombinant proteins in tongue sole were 0.3 mg/mL and 0.25 mg/mL, respectively. The results of Western blot and mass spectrometry analysis indicated that the obtained recombinant proteins of LepA and LepB had correct sequences and immunological activity. The proteins were purified by Ni2+-NTA affinity chromatography, and high-purity recombinant proteins of LepA and LepB were obtained. In vitro incubation of hypothalamus with recombinant LepA and LepB proteins from C. semilaevis indicated that they could significantly inhibit the expression of endogenous lepa, lepb and gnrh3 mRNA, which verified that the obtained recombinant proteins have biological activities. The results could help exploration of the physiological role and regulation mechanism of leptin in growth and development of C. semilaevis.
Great blue-spotted mudskippers (Boleophthalmus pectinirostris) have lived in mudflats along the coasts of Japan and South China for a long time. Compared with the fish living in water, B. pectinirostris, as an amphibious fish, is more vulnerable to terrestrial and aquatic pathogenic factors. Interleukin-8 is a CXC chemokine that plays an important role in regulating the inflammatory response. In this study, we screened the interleukin-8 gene from skin transcriptome of B. pectinirostris, then cloned and verified its coding region. The results indicated that the open reading frame(ORF) of B. pectinirostris IL-8 gene was 306 bp, coding 101 amino acids, including a typical signal peptide of 18 amino acids, and a SCY domain of 62 amino acids, which also possessed the four conserved cysteine residues, namely Cys-30, Cys-32, Cys-57 and Cys-73. Like most fish, B. pectinirostris interleukin-8 amino acid sequence lacked the ELR motif which was the receptor binding site and it was replaced by Asn-Ser-His (NSH) in great blue-spotted mudskipper. In all known fish amino acid sequences, rules of this motif were as follows: the first amino acid was never the amino acid with positive charge, the second amino acid was generally those amino acids with no charge, and most of third amino acid were amino acids with positive charge. Phylogenetic analysis showed that purification selection affected the diversity of this motif in fish. Tissue expression analysis by RT-qPCR indicated that interleukin-8 gene was widely expressed in healthy tissues of B. pectinirostris and it had the highest expression in the gill and brain. Bacteria and poly (I∶C) injection experiments showed that expression of IL-8 in liver, spleen and brain tissues was up-regulated after the infection, indicating that IL-8 plays an important role in the inflammatory response and immune response of liver, spleen and brain tissues. This study provided important reference for the research of immune genes of B. pectinirostris, thus having more comprehensive understanding of the evolution process and function of fish immune genes.
The study was carried out to investigate the effect of dietary selenium(Se) supplementation on the growth performance, serum antioxidant enzyme activity and expression of related genes in liver of juvenile turbot (Scophthalmus maximus) under copper(Cu) stress. Four experimental diets(D1,D2,D3,D4) were formulated to contain the following diets with increment levels of copper 0, 1 000, 1 000, 1 000 mg/kg and selenium 0, 0, 2, 4 mg/kg. The group D1 as the control. Each diet was randomly assigned to triplicate groups of 40 fish [initial weight, (24.85±0.1) g] per aquarium. Fish were fed twice daily(8:30 and 16:30) at ratio of 1.5% or 2% body weight for 84 days. Results showed that the different diets had no significant effect on the survival rate of turbot. The weight gain rate, specific growth rate and protein efficiency ratio of D2 group were significantly lower than other groups. Whereas, feed conversion ratio and feed intake reached the maximum in group D2. Crude lipid content of whole body and liver all showed a trend of first decreasing and then rising, and, there were no significant differences of crude lipid and crude protein contents in muscle among all groups. Cu concentration of D2 group in whole body, vertebra and liver was remarkably higher than the others. Zn concentration in whole body, vertebra and liver reached the maximum in the group D4. Hepatic Fe concentration was lower in group D4 than the others. The serum total antioxidant activity, catalase activity, glutathione peroxidase activity decreased in group D2, and increased with further increase of selenium level. While the malondialdehyde content followed the opposite trend. Total superoxide dismutase and CuZn superoxide dismutase activity both reached the minimum in group D4. Both alanine aminotransferase and aspartate transaminase activities showed their highest values in group D2, which were significantly higher than those in the other groups. Glucose, triglyceride, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol showed a opposite trend to alanine aminotransferase and aspartate transaminase activities with the lowest value in group D2, significantly lower than that in the control group. Metallothionein expression increased in the liver with the dietary selenium levels under Cu stress, which was significantly higher in group D4 than that in the other groups. Glutathione transferase and lysozyme expression levels decreased initially and then increased with the dietary change, which were significantly lower in group D2 than those in the control group. Heat shock protein 70(HSP70) expression level showed the highest values in group D2, which was significantly higher than those in the other groups. In conclusion, adequate dietary selenium levels could affect the growth performance, increase the total antioxidant capacities, modulate some mRNA expression of gene in liver, and then reduce the metabolic disorders caused by high copper content in juvenile S. maximus.
C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense (MnLec3) was obtained using rapid amplification of cDNA ends method (RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRT-PCR. The full-length cDNA sequence of MnLec3 was 1 357 bp, which contained a 5′ untranslated region of 125 bp, a 3′ untranslated region of 206 bp, a 1 026 bp open reading frame (ORF) encoding 341 amino acids. The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.
Spermatogenesis is the process in which spermatogonia develop into mature spermatozoa. In most animals, this process involves changes in the basic protein associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines with strong alkalinity. With the replacement of basic proteins, nuclei progressively undergo chromatin condensation. The Chinese mitten crab (Eriocheir sinensis) sperm nucleus chromatin arrange loosely, and its formation is not clear. In this research, DNA sequence encoding histone H2A was cloned by PCR method. Thereafter, polyclonal antibody against the histone H2A was generated in rabbit, histone H2A distribution during E. sinensis spermatogenesis was detected through immunofluorescence. The encoding sequence of E. sinensis H2A contains 369 bp that encodes a protein of 123 amino acids. The predicted molecular weight of H2A was 13.1 ku. Protein alignment studies demonstrated that this histone H2A had a high identity with its counterparts in Penaeus vannmei and P.monodon, and the homology both are 99.19%. Immunofluorescence demonstrated that histone H2A was present through the spermatogenesis of E.sinensis. In spermatogonia and spermatocytes, H2A was mainly distributed in the nuclei and cytoplasm, while in spermatids and spermatozoa, H2A was mainly in the nuclei. The persistence of H2A in the mature sperm nucleus of E. sinensis might have some association with the uncondensed chromatin.
Calcium/calmodulin-dependent protein kinase Ⅳ (CaMKⅣ) which has multiple physiological functions in organisms, is a regulatory factor widely found in eukaryotes. To investigate the role and molecular mechanism of CaMKⅣ in Onchidium struma in the process of evolution from sea to land, and further explain the physiological function of CaMKⅣ to provide a scientific theoretical basis, a series of experiments have been carried out. In this study, the cDNA sequence of CaMKⅣ gene was cloned by RACE-PCR using ganglion of O. struma, and bioinformatics analysis and qRT-PCR experiment were performed. The results suggested that the full-length cDNA of CaMKⅣ gene consists of 1 603 base pairs (bp), 315 bp 5′ non-coding region (UTR), 256 bp 3′ non-coding region (UTR) and 1 032 bp open reading frame (ORF) which encode a total of 343 amino acids. The atom of the polypeptide chain encoded by this gene was predicted. The CaMKⅣ protein was predicted to contain 5 490 atoms, the molecular weight is about 3.87 ku, the theoretical isoelectric point is 6.12, the molecular formula is C1 741H2 768N452O514S15, and the N-terminal signal peptide is composed of 1-29 amino acids. The amino acid sequence alignment and phylogenetic tree results suggest that the CaMKⅣ gene was the closest to the CaMKⅣ gene of Aplysia californica and Biomphalaria glabrata, and its phylogenetic analysis was consistent with the traditional morphological classification. The results of qRT-PCR indicated that the CaMKⅣ gene was expressed in all tissues, and the relative expression in the pleopod was the highest, followed by the dorsal skin and mouthparts, and the expression level in the ganglion, albumen gland, intestine, hepatopancreas and other organs were low. It was preliminarily speculated that the gene played an important role in the ganglion of O. struma, or it was the molecular basis for the biological body to perceive external environmental variables. The results of this experiment will provide a theoretical support for further understanding of the regulation of the physiological functions of nervous structure and the function of CaMKⅣ gene, and also provide a reference for exploring the environmental adaptation mechanism of marine animals in the process of evolution from ocean to land.
Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein in the Toll-like receptor (TLR) signaling pathway and plays an important role in signal transmission. In this study, the full-length cDNA sequence of a MyD88
Pacific saury (Cololabis saira) is one of the most important commercial pelagic fish species being harvested in the Northwestern Pacific Ocean region. Spatial and temporal dynamic monitoring of fishing boats has become an important data source for understanding the distributional dynamics of fisheries. Visible infrared imaging radiometer suite (VIIRS) Day/night band (DNB) night-time remote sensing images can be used to monitor night fishing boat lights. In this study, peak detection and threshold segmentation techniques were used to identify fishing boats employing DNB images in the Northwest Pacific. GIS tools were used to extract and analyze the fishing boat position and numbers. The fishing log of C. saira resources and the selected NPFC fishing boats list were used to validate the identified results. The results show that the method proposed in this study can effectively identify the location and operation status of C. saira fishing boats, and provide useful information for understanding the spatial distribution and dynamics of Pacific saury fishing zones in the northwest Pacific region. Sea surface temperature data provided by the National Oceanic and Atmospheric Administration (NOAA) were used to analyse temperature changes in the fishing ground of C. saira. The results showed that the SST range of lighting fishing boats changes with the migration of saury. From July to September 2016, the range of fishing ground SST fluctuated greatly because Pacific saury are widely distributed, making extensive migrations from subtropical to subarctic regions throughout the Kuroshio-Oyashio Currents transition zone. After September, the SST of the fishing ground tended to be stable. In the future, this information will be useful to forecast the potential fishing zone and to assess the fishing resources.
According to the yield data of the Sthenoteuthis oualaniensis in the southern and northern sea areas of the South China Sea in spring in 2015 and 2016, the present research was carried out to analyze the relationship between depth, working time and moon phases and CPUE under the condition of operation. From the GAM model analysis, we knew that the effect of operation time and lunar day on CPUE was significant. The results showed that CPUE is relatively low at the depth of 0-999 m under the water in the bright moonlight. When there was nomoonlight, the CPUE in each water layer changed little, and the CPUE at the depth of 2 500-2 999 m was relatively higher. When the working time in the first half of the night (21:00—24:00), the CPUE was higher, which wan up to 399.4 kg/net at most, and the CPUE gradually decreased in the second half of the night (0:00—5:00). There was a great difference in the yield between the bright moonlight and moonless night, which were respectively 267.0 kg/net and 321.6 kg/net. El Niño has impact on the changes of the S. oualaniensis resources in various water layers to a certain extent. The research on the effect of operational conditions on CPUE of the S. oualaniensis can provide reliable suggestions for fishing ground.
To investigate the effects of dietary baicalein on the growth performance, anti-oxidation indicators and flesh quality of grass carp, five diets were prepared with the baicalein addition at 0 (control diet), 0.1, 0.2, 0.4, 0.6 g/kg, and fed to grass carp with an initial body weight of (75.8±0.24) g. After 60 days of feeding, the weight gain showed quadratic relationship with dietary baicalein level, and the supplementation of 0.2 g/kg baicalein improved weight gain by 8.63% and decreased feed conversion ratio by 0.14 when compared to the control group. Serum SOD activity increased and MDA level decreased by the addition of 0.2−0.6 g/kg baicalein. Serum CAT activity also increased by the addition of 0.4 g/kg baicalein, while AKP and lysozyme activity showed no significant difference among groups. The addition of 0.4 and 0.6 g/kg baicalein increased the total amino acids and the total essential amino acids in flesh, and the total nonessential amino acids increased by the addition of 0.6 g/kg baicalein. There were no significant differences in the proximate composition and water-holding capacity of flesh, and muscle fiber density and diameter also showed no difference among groups. In conclusion, dietary baicalein could improve the growth and anti-oxidant ability of grass carp, and the recommended supplemental level of baicalein was 0.2 g/kg.
In order to investigate the effects of dietary sodium selenite and selenoyeast on growth performance, antioxidant responses and low temperature stress resistance of juvenile yellow catfish Pelteobagrus fulvidraco, four isonitrogenous and isolipid diets containing graded levels of Se, 0.028 (control, G0), 0.25 (sodium selenite, G1), 0.30 (selenoyeast, G2), 0.52 (selenoyeast, G3) mg/kg were randomly fed to P. fulvidraco [initial weight (2.12±0.01) g] for 56 days. At the end of breeding experiment, the low temperature stress test was conducted. The results showed that sodium selenite and selenoyeast had no significant effects on the final weight, weight gain rate, feed coefficient and survival rate. Sodium selenite and selenium yeast significantly increased the selenium content of whole body and muscle, and G3 group was significantly higher. The selenium content of whole body had no significant difference between G1 and G2 groups, but the selenium content of muscle in G2 group was significantly higher than that of G1 group. Sodium selenite and selenium yeast significantly increased GPx activity in liver. In G1 and G2 groups, SOD activity was significantly higher than that in G0 group. At 22 °C, the total protein, cholesterol, triglycerides, and urea nitrogen contents had no significant difference among all the groups, but G2 group had significantly higher glucose than G0 and G1 groups. The relative expression of HSP70 mRNA had no significant difference. At 13 °C, the total protein, cholesterol content had no significant difference. The triglyceride content in G1 group was significantly higher than those in G0 and G3 groups, but was not significantly different compared to G2 group. Glucose in G2 and G3 groups were significantly higher than those in G0 and G1 groups. The urea nitrogen content in G2 group was significantly lower than that in G1 group. The relative expression of HSP70 mRNA in G0 group was significantly higher than those in other groups. The results showed that 0.30 mg/kg selenoyeast in juvenile P. fulvidraco diet was better than 0.25 mg/kg sodium selenite, taken the growth performance, selenium deposition, antioxidant activity and low temperature stress resistance into consideration.
In aquaculture, high fat diets are widely used for its protein sparing effect which can improve protein utilization efficiency, promote growth and protect environment pollution. Hoevever, it can also lead to excessive lipid deposition of liver and lower disease resistance. In order to solve this problem, additives should be explored. Tea polyphenols are natural plant extracts which have been proved to have the effects of of promoting growth, lipid reduction and immunity improving in many animals. Therefore, the purpose of the experiment is to investigate the effects of dietary tea polyphenols (TP) level on growth, antioxidant capacity and lipid metabolism of turbot. Four diets with graded levels of TP (0.00%, 0.01%, 0.02% and 0.05%. dry weight 0, 100, 200 and 500 mg/kg) was formulated in this experiment. Fish were cultured in indoor aquaculture system, and each diet was randomly assigned to three replicate groups of juvenile fish [initial average weight of (13.51+0.31) g] and fed twice daily (8:00, 18:00).The feeding trial lasted 70 d. The results showed as follows: ① Compared with the control group, fish fed the diet with 0.01%–0.02% TP had significantly higher weight gain rate (WGR). Feed efficiency (FE) was increased with the increasing TP level and no significant differences were detected among dietary treatments. Compared with the control group, Hepato-somatic index (HSI) was significantly increased by the supplement with TP. ② The lipid contents of whole fish and liver were decreased with increasing TP level and significantly lower in 0.02%–0.05% group compared with the control group. ③ Compared with the control group, the activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) were significantly higher in fish fed the diet with TP, and no significant differences were detected among fish fed diets with TP. The activity of superoxide dismutase (SOD) first increased and then decreased with the increasing TP level, and 0.02% supplement level was the highest. The content of malondialdehyde (MDA) was decreased with increasing TP level, and significantly higher in 0.02% group compared with the control group. ④ The expression of sterol regulatory element binding protein 1(SREBP-1) was decreased with increasing TP level and significantly higher in 0.02%–0.05% groups compared with the control group. The expression of fatty acid synthetase (FAS) was first decreased and then increased with increasing TP level and significantly lower in 0.02% group compared with the control group. The expression of peroxisome proliferator-activated receptor α (PPAR α) first increased and then decreased with increasing TP level and significantly higher in 0.02% group compared with the control group. The expression of carnitine palmitoyl transferase 1(CPT 1) increased with increasing TP level and significantly higher compared with the control group. This research indicated that high fat diets supplemented with TP could promote growth, reduce liver fat deposition, and increase antioxidant activity. The optimum supplemental level of TP in diets for turbot is approximatly 0.02%.
The basic nutrients, amino acid composition and mineral element content of seven marine fishes were determined, and the amino acid was evaluated for nutrition. At the same time, the upper limits (UL) of nutrients were used to assess the mineral elements in fish muscles. Health risk analysis. The results showed that there were no significant differences in the crude ash content of the seven marine fishes, and the crude fat and water contents were significantly different. Larimichthys polyacits had the highest crude fat content and the lowest water content, which were 9.44%±0.29% and 71.64%±0.44%, Scarus ghobban had the highest water content, the lowest crude fat content, and the crude protein content was between (18.56%±0.29%) and (20.97%±0.32%), the difference was significant; seven marine fishes amino acid species were complete, and the ratio of ∑EAA/∑TAA and ∑EAA/∑NEAA is in line with the FAO/WHO ideal model, with the limiting amino acids Val, Trp, Met+Cys, and the essential amino acid index between 77.57 and 89.04; 7 tested mineral elements in fish muscle were rich, and the content of the same elements in different fish species was significantly different. The average content of K element was the highest and the difference of Na element content was the most significant. The risk assessment of mineral elements showed that Zhuhai sampling had the maximum risk index of Cu in fish in the age groups of 2-5, 6-17, 18-44, 45-59 is greater than 100%, which is an unacceptable risk range, and the average risk index is within the acceptable range. The above results may provide a reference for the deep processing of marine fish and the daily diet of residents.
In this study, three cooking methods of boiled, fried and braised shrimps were used to treat the meat of Litopenaeus vannamei, and the cooked shrimp was subjected to in vitro simulated saliva-gastric-intestinal digestion to investigate the immunological activity after cooking. Changes of major allergen tropomyosin in shrimp were analyzed by SDS-PAGE, Western-blotting and indirect ELISA. The results of SDS-PAGE showed that there was no significant change in the TM bands by three cooking methods, indicating that the TM did not undergo degradation during cooking; the results of immunoblotting and indirect ELISA showed that the immunological activity of TM in boiled, fried and braised shrimp lost 2.08%, 11.33% and 15.56%, respectively. However, the immunological activity of the in vitro simulated digestion products of shrimp meat after cooking was significantly lost, and the immunological activitiy of boiled, fried and braised shrimp were decreased by 86.90%, 88.94% and 97.39%, respectively. Studies have shown that the reduction of shrimp TM immunological activity mainly occurs in the simulated intestinal fluid digestion stage, three cooking treatments can reduce the immunological activity of TM, and the braising treatment significantly reduced the immunological activity of TM. This experiment provides a theoretical basis for the study of allergen reduction mechanism and the development of hypoallergenic aquatic products.
The high density polyethylene (HDPE)/starch composites were prepared by melt blending. The effects of starch contents on the rheological properties, structure, thermal properties, mechanical properties and dynamic mechanical properties of HDPE/starch composites were studied. The results showed that the introduction of starch improved the rheology and toughness of the composites. When the content of starch was 20 wt%, it was uniformly dispersed at micron -scale thickness in HDPE/ starch composites. When the content of starch increased to 30 wt% and 50 wt%, the starch exhibited spherical dispersed phase in HDPE/starch composites, and the average diameter of starch increased from 0.6 μm to 0.8 μm. The breaking strength and breaking elongation of HDPE/ starch blends decreased significantly with the increase of starch content. All the blends with different starch contents were detected to three transition peaks. The γ transition of HDPE at low temperature corresponds to the glass transition peak of polyethylene, it was related to the movement of the amorphous chain segment, and the content of starch had little effect on the γ transition. The β transition was related to starch phase, there was no β transition for pure HDPE, and the peak became wider with the increase of starch content. The α transition in high temperature zone was related to the movement of the restricted chain near the crystallization zone of polyethylene, and it was a complex multiple relaxation process. Introducing a certain content of starch can effectively enhance the rheology and toughness of the composites, and this result can provide a theoretical and technical basis for the high- performance fishing materials based on degradable starch.