Abstract:

Abstract:

Abstract:This research was conducted based on the mix ratio of ordinary concrete with compressive strength of C25, with the aim to use scallop shells to partly replace coarse and fine aggregates in concrete. Experiments were conducted on the mix ratio of scallop shell concrete, and the working performance and basic mechanical properties of coarse and fine scallop shells instead of natural aggregate concrete was compared. According to the reef plan of Shandong Province from 2016 to 2020, the carbon sequestration potential of the natural aggregate in the artificial reef concrete replaced by the scallop shells and the accumulation area of the cleanable waste scallop shells could be estimated. The results showed that as the proportion of scallop shells replacing natural aggregate increases, the collapse of concrete tends to decrease, and the slump of natural coarse aggregate concrete replaced by coarse scallop shells is larger than that of natural fine aggregate concrete replaced by fine scallop shells. When scallop shells replaced natural stone in concrete, the compressive performance of scallop shell concrete was similar to that of the base concrete. The optimum replacement rate of scallop shells as coarse aggregate was 40%, and the 28-day compressive strength of scallop shell concrete was 7.3% higher than that of the reference concrete. When the natural fine aggregate in concrete was replaced by scallop shells, the compressive performance of concrete was greatly improved relative to that of the reference concrete. The best replacement rate was 60%, and the 28-day compressive strength of scallop shell concrete was 33.5% higher than that of the reference concrete. Estimation replacement rate of fine aggregate with scallop shells for concrete was 50%~70%. For example, 20.43 million empty artificial reefs planned for 2016~2020 in Shandong Province are all made of this kind of concrete, reef materials can achieve carbon sequestration of 275.4~ 385.6 thousand tons and reduce the land accumulation area of abandoned shells by 5.89~8.24 square kilometers, which has high ecological value.

Abstract:In this study, we used a fuzzy synthetic evaluation to build an ecosystem-health assessment model according to offshore sea ecosystem characteristics and artificial reef construction functions from three aspects: Physicochemical environment, biological community structure and ecosystem function. To eliminate the dimension effect of sub-indexes, normalization process was used. An analytic hierarchy process was used to assign a threshold or the ecological indicator. The synthetic health index was used to assess the health status of the artificial reef areas, radiation areas and contrast areas. Sub-indexes of artificial reef ecosystem health assessments included physical, chemical, and biological factors. Assessment standards were based on seawater quality, standard marine sediment quality, and guidance for the assessment of coastal marine ecosystem health. We conducted a series of ecosystem health assessments of artificial reefs, and radiation and control areas of Danglang Island based on the survey results of 5 voyages in 2013~2015. The results showed that the ecosystem of artificial reef areas was in a healthy state, whereas the control area was in a sub-healthy state. From the highest to the lowest, the ecological health index was: The three reef area (0.783)>radiation areas (0.646)>control area (0.594). Compared with non-reefing, the ecosystems of the reef and radiant areas were all separated from the sub-healthy state and gradually improved, however, the control area was still in a sub-healthy state and its development was unstable. Therefore, the conclusion was that the ecosystem of the artificial reef areas of Danglang Island were more stable than the control areas, and ecosystem health of radiation areas, was gotten better, due to effect of reefs. The study of reef ecosystem health and scientifically assessing artificial reef ecological effects is important for the construction of reef projects, environment protection, stock enhancement, efficient administration, and sustainable development of fisheries.

Abstract:The double-bottom cages for flounder fish tend to move and deform under waves. Therefore, it is necessary to carry out hydrodynamic analysis for the stabilization of flounder fish cages. Herein, a numerical model of deformation of double-bottom cages under waves was established based on the finite element method, and then maximum displacement and the pitch of the double-bottom were calculated. First, results of the upper bottom were compared with that of the lower bottom. Afterwards, the results of the lower bottom of double-bottom cage were compared with that of the single-bottom cage. The simulation results showed that the two bottoms of double-bottom cages were able to stay relatively parallel during wave periods. The maximum displacement and pitch of the two bottoms increased along with not only wave height but also wave period. In addition, the inclined direction of the two bottoms was the same. Under the same wave conditions, the horizontal of displacement and the maximum pitch of lower bottom were larger than those of the upper bottom, but their vertical displacements were not much different. It was found that the difference in pitch between upper and lower bottoms was the largest when the wave height was 15 cm and the wave period was 1.4 s. However, the upper and lower bottoms of the cage did not collide with each other, and the two bottoms of the cage could remain relatively stable. Moreover, under the same wave conditions, the displacement of the lower bottom of the double-bottom cage was less than that of the single-bottom cage, but the pitch of the lower bottom of the double-bottom cage was larger than that of the single-bottom cage. Additionally, the maximum mooring-line force of double-bottom cages for flounder fish was larger than that of single-bottom cages. Furthermore, the study on the hydrodynamic characteristics of double-bottom cages for flounder fish under the combined action of wave and flow should be carried out in the future.

Abstract:The present study aimed to quantitatively evaluate the ecological effects of artificial reefs from the perspective of the food-web structure and provide reference material for marine ecological restoration and conservation research. A stable isotope technique was applied to analyze consumers and their potential carbon sources. Samples were collected from the reef areas built in 2010, 2012, and 2014 and the control area in Dashentang during June 2016. An IsoSource model was used to construct the food web. Stable isotopes were used to calculate the trophic levels of the consumers. The results showed that food sources can be categorized into three categories based on the δ13C values of phytoplankton, particulate organic matter, and soil organic matter. Phytoplankton represented the largest source of carbon for consumers (67.2%~81.5%). The findings from the IsoSource mix model revealed that phytoplankton were the main contributors to the food web in the studied areas. There were no significant differences in the δ13C or δ15N values of the same food sources from different areas. The δ13C values of suspension- feeding bivalves, including Arca subcrenata, Ruditapes philippinarum, and Crassostrea gigas, were significantly higher in the reef areas than in the control area. The proportion of phytoplankton in the carbon sources for suspension-feeding bivalves was significantly higher in the reef areas than in the control area. The δ15N values of consumer species ranged from 7.70‰ to 14.34‰, and the trophic levels ranged from 2.0 to 3.95. The δ15N values or trophic levels of piscivorous consumers were higher in the reef areas than in the control area, and the level of significance increased with the duration of the reefs. The findings indicate that artificial reefs might induce an abundance of organisms in reef areas, leading to changes in the composition of the food sources of suspension-feeding bivalves and an increase in the trophic levels of piscivorous consumers.

Abstract:In the natural environment, there is reproductive isolation between Epinephelus tulcula and E. fuscoguttatus due to their different geographical distribution and reproduction time and temperature. In this study, we used frozen sperm from E. tulcula and eggs from E. fuscoguttatu for artificial insemination. The fertilized eggs were incubated in water at a temperature of 30℃ and salinity of 20. The embryonic development and post-embryonic metamorphosis were observed in detail. The morphological characteristics and developmental times of hybrid offspring were observed using a microscope and dissecting mirror. We determined the fertilization rate, hatching rate, and deformity rate, and the fry growth parameters were measured. The results showed that the fertilized eggs completed embryo development 1037 maf (min after fertilization). Early in gastrula an embryonic shield was formed on one side of the germ layer, and the neural tube also began to appear when the blastopore was closed. Additionally, as the optic vesicle and the ear vesicle developed, the Kirschner's capsule (end ball) also developed. It was found that the embryoid twitch preceded the heartbeat. When the sarcomere developed to 23 pairs (977 maf), the heart began to beat intermittently at a rate of 98 beats/min and the interval of 24~40 s. The interval of the heartbeat reduced at 1018 maf with a heart rate of 93 beats/min, and then the intermittent phenomenon disappeared. These phenomena have not been documented in previously. The typical feature of metamorphosis is the growth and degeneration of the trigeminal spine. When the hybrid juvenile grew to 3 mm (10 daf, days after fertilization), it formed dorsal and pelvic pin spines. When the length reached 21.5 mm (35 daf), the trigeminal spine degenerated and the metamorphosis was complete. When hybrid juveniles grew to 55.3 mm (45 daf), their phenotypic characteristics were similar to that of the adult. The fertilization rate, hatching rate, and malformation rate of the frozen sperm were (76.67±5.77)%, (85.67±5.13)%, and (6.33±1.54)%, respectively, and there was no significant difference in these parameters when compared to those of fresh sperm (P<0.05). At the 65 daf, the body length of hybrid fry reached 8.89 cm. Then, the offspring of the hybrids tended to be more consistent in body color and they grew very fast. The average body length and body weight of hybrid fish were (32.58±1.60) cm and (559.76±80.85) g at one year old. By contrast farming, the weight of E. fuscoguttatus♀ × E. tulcula ♂ was 1.63-fold of E. fuscoguttatus♀ × E. lanceolatus♂, and the total length of E. fuscoguttatus♀ × E.tulcula♂ was 1.24-fold of E. fuscoguttatus♀ × E. lanceolatus♂. The growth equation in this period was W=0.0311L2.8079, R²=0.9886. The results suggest that there is no genetic disorder in the hybrid embryos and post-embryonic development between E. tulcula♂ and E. fuscoguttatus♀. The offspring showed high fertility and hatching rate, but low malformation rate. Moreover, the offspring showed heterosis of rapid growth. The study provides a genetic basis for hybrid breeding and seed cultivation of these two grouper species.

Abstract:Artificially induced gynogenesis is a form of chromosome manipulation for sex control, accelerating both the elimination of recessive deleterious genes and the rapid establishment of inbred lines that could benefit the breeding progress in fish species. Artificially induced gynogenesis can be divided by mechanism into meiogynogenesis and mitogynogenesis. In this study, the fourth generation of a successive meiogynogenetic population (G4) in Japanese flounder (Paralichthys olivaceus) was established by induction of meiogynogenesis, in which the eggs of third-generation females (G3) were activated by ultraviolet-irradiated red sea bream (Pagrus major) sperm, followed by a cold-shock treatment at 0℃, starting 3 min after activation and lasting 45 min. We evaluated the genetic structure of the control, G2, G3, and G4 populations with 24 microsatellite markers and a high recombination rate that covered all linkage groups of the flounder. The efficiency of successive meiogynogenesis in terms of producing a highly inbred line was quantified by calculating the homozygosity and genetic similarity. The results showed that 96, 42, 32, and 32 alleles were detected in the control, G2, G3, and G4 populations, respectively; the average numbers of alleles were 4.00, 1.98, 1.33, and 1.33, respectively; and the average expected heterozygosities were 0.6416, 0.3472, 0.1694, and 0.1492, respectively. The average homozygosities among the 24 analyzed loci were 0.3503, 0.6528, 0.8306, and 0.8508, respectively. In the G4 population, 17 loci were homozygous, while 7 remained heterozygous. With respect to genetic similarity, the average similarity indexes between offsprings within populations were 0.5822, 0.9238, 0.9890, and 0.9988, respectively. Among G1, G2, and G3 populations, the homozygosity and genetic similarity index increased significantly (P<0.05) with generations. However, the differences in homozygosity and genetic similarity index between G3 and G4 were not significant. Our results indicate that artificially induced successive meiogynogenesis can effectively increase the homozygosity of individuals, as well as the genetic similarity of offspring within a population. Successive meiogynogenesis has a higher induction rate than mitogynogenesis, and therefore is a useful alternative method for establishing clonal lines in fish.

Abstract:This study was conducted to evaluate the effects of replacing fish meal with composite protein on growth performance, diet digestibility, and gut health in Japanese seabass (Lateolabrax maculatus). Five isonitrogenous and isolipidic diets were formulated by replacing 0, 20%, 40%, 60%, or 80% of fish meal in the basic diet with composite protein. Quadruplicate groups of 30 fish [initial body weight (5.80±0.07) g] were fed the test diets to apparent satiation twice daily (08:00 and 16:30), and the experiment continued for 70 days. During the feeding trial, water temperature, salinity, and dissolved oxygen values were maintained at (29.5±1.5)℃, 29±1, and ≥6.5 mg/L, respectively. Samples were collected for analysis after the feeding trial. The main indexes including the growth performance, diet digestibility, intestinal digestive enzymes activities, posterior intestine morphology structure, and expression of inflammation-related genes of the proximal intestine, mid-intestine, and posterior intestine were analyzed to investigate the appropriate replacement proportion of fish meal by composite protein. The results showed that substituting 20% of fish meal with composite protein did not significantly affect growth performance compared to that of the control group (P>0.05), while further increment of fish meal replacement levels resulted in significant reduction of weight gain, survival rate, feed efficiency, and protein efficiency ratio (P<0.05). Moreover, as the replacement level of fish meal with composite protein exceeded 20%, intestinal protease activity and apparent digestibility coefficients of feed dry matter, crude protein, and crude lipid also significantly decreased with increasing level of fish meal replacement by composite protein (P<0.05). Meanwhile, increasing replacement of fish meal with composite protein damaged posterior intestine morphology structure and up-regulated expression of gut inflammatory genes such as TNF-α and IL-1β, while an opposite trend was observed for the expression of anti-inflammatory genes, IL-4 and IL-10. In conclusion, it was recommended that the replacement proportion of fish meal with composite protein should not exceed 20%.

Abstract:An 80-day feeding trial was conducted to investigate the pantothenic acid dietary requirements of juvenile turbot. Six isoenergetic and isonitrogenous diets were formulated using fish meal and casein as a protein source. The basal diet was supplemented with calcium D-pantothenic acid at 6.24, 10.64, 15.02, 23.81, 41.40, or 76.57 mg/kg and fed to juveniles weighing (24.73±0.10) g. The results were as follows: 1) No significant differences in juvenile turbot survival rate (SR) were found between the dietary treatments (P>0.05) and the weight growth rate (WGR) and specific growth rate (SGR) increased as pantothenic acid levels increased from 10.64 to 76.57 mg/kg (P<0.05). For dietary pantothenic acid content greater than 23.81 mg/kg, the hepatosomatic index decreased significantly (P<0.05). 2) The crude protein and lipid levels of the whole body and crude protein levels of the muscle initially increased and then decreased with increasing dietary pantothenic acid levels, whereas the crude lipid content of the liver decreased (P<0.05). 3) The activities of the intestinal digestive enzymes Na+, K+-ATPase and hepatic cholinesterase (ChE) initially increased and then decreased with increasing dietary pantothenic acid levels, whereas intestinal creatine kinase (CK) activity increased significantly from 10.64 to 76.57 mg/kg (P<0.05). 4) Hepatic and serum catalase (CAT) and superoxide dismutase (SOD) activities were significantly lower in the control group than in the groups with calcium pantothenic acid-enriched diets (P<0.05). Turbot fed a 6.24 mg/kg pantothenic acid diet had a higher serum malondialdehyde content than those fed other diets (P<0.05). 5) Fatty acid synthetase (FAS) expression increased and then decreased with increasing levels of pantothenic acid. Lipoprotein lipase (LPL) expression was significantly up-regulated in the liver with increasing levels of pantothenic acid (P<0.05). In conclusion, appropriate levels of dietary pantothenic acid significantly improved intestinal digestion and absorption capacity, thus, improving nonspecific immunity and the expression of fat-related genes, and consequently, the growth and body composition of juvenile turbot. Based on broken-line regression analysis of WGR, the optimum dietary pantothenic acid requirement of juvenile turbot with a body weight of 24.73 g was 16.08 mg/kg.

Abstract:To investigate the effects of lysozyme as a green feed additive on the development of the digestive tract and digestive utilization of nutrients in GIFT tilapia, a 60-day feeding trial was conducted with graded levels of dietary lysozyme (0, 18, 36, 54, 72, and 90 mg/kg, marked as L0, L18, L36, L54, L72, and L90 respectively). The results were as follows: The fish had a different feedback response on the digestive enzyme activity in the liver and the gastrointestinal tract among groups; the protease activity in the stomach, and the anterior and middle intestine of L36~L72 groups was significantly higher than that of the control group (P<0.05); the lipase activity in the liver and the anterior intestine of the L54 and L72 groups was significantly higher than that of the control group (P<0.05); and the amylase activity in the digestive tracts (anterior and middle intestine excluded) of the L72 and L90 groups was significantly higher than that of the control group (P<0.05). The intestinal morphology showed that the values of villus density, villus height, and villus width of different intestinal parts in L36~L72 groups were higher than those of the control group, while the muscular thickness of the anterior and distal intestine was reduced in dietary lysozyme groups. The thickness of the middle intestines of the L18, L36, and L90 groups was significantly lower than that of the control group (P<0.05), and there was a firstly increased and then decrease tendency. The thickness of the middle intestines of L36~L72 groups were all higher than the control group with no significant difference (P>0.05). The goblet cell numbers were more in the L54 and L72 groups than in the control group (P<0.05). The liver morphology showed that the liver cells were more voluptuous and denser in the L36 and L54 groups than in the control group, while worse health condition was found in the L90 group. With regards to apparent nutrient digestibility, the crude protein digestibility was significantly higher in the L36 and L540 groups than in the control group in periods I and Ⅱ (P<0.05), and in periods Ⅲ and Ⅳ. The digestibility of dry matter, crude protein, and crude lipid were significantly higher in L36~L90 groups than in the control group (P<0.05). The results above indicated that 36 and 54 mg/kg dietary lysozyme had the most stable efficacy, which could improve GIFT tilapia dry matter, crude protein, and crude lipid digestibility by promoting liver and intestine development and digestive enzyme activity.

Abstract:In this study, we examined the biological toxicity of polybrominated diphenyl ethers, new persistent organic pollutants that can accumulate in organisms and have toxic effects in higher nutritional levels of the food chain. We performed acute toxicity tests using four polybrominated diphenyl ethers (PBDE) congeners, BDE-47, BDE-99, BDE-153, and BDE-209, on three species of marine organism, Chlorella pyrenoidosa, Daphnia magna, and Scophthalmus maximus. The 96 h concentrations for 50% maximal effect (96 h EC50) and 50% lethality (96 h LC50) were calculated for each species. The 96 h LC50 and safe concentrations of the four PBDE homologs on C. pyrenoidosa were as follows: BDE-47, 1.39 and 0.10 μg/L; BDE-99, 1.76 and 0.18 μg/L; BDE-153, 3.23 and 0.27 μg/L; and BDE-209, 378.62 and 8.68 μg/L, respectively. The 96 h LC50 and safe concentrations of the four PBDE homologs on D. magna were as follows: BDE-47, 0.24 and 0.03 μg/L; BDE-99, 1.42 and 0.82 μg/L; BDE-153, 1.49 and 0.94 μg/L; and BDE-209, 63.93 and 5.09 μg/L, respectively. The 96 h LC50 and safe concentrations of the four PBDE homologs on S. maximus were as follows: BDE-47, 5.46 and 2.02 μg/L; BDE-99, 6.07 and 2.01 μg/L; BDE-153, 7.35 and 2.17 μg/L; and BDE-209, 118.78 and 58.62 μg/L, respectively. BDE-47 was shown to be particularly toxic to marine organisms, with toxicity decreasing according to the number of Br atoms in the congener molecule in the following order: BDE-47, BDE-99, BDE-153, and BDE-209. The PBDE content of global seawater is currently at the pg/L level, whereas the 96 h EC50 and 96 h LC50 of the four homologs was at the µg/L level, much higher than the PBDE content of the marine environment. However, owing to the structural stability and environmental persistence of PBDEs, their effects on environmental health cannot be ignored, and further studies will be conducted on their toxicity.

Abstract:DNA methylation is one of the most important epigenetic modifications. It is vital to maintain the stability of the whole genome and regulate the expression of tissue-specific genes. Methylation level and distribution throughout genomic DNA are significantly correlated with gene expression rate. In order to investigate the changes of genomic DNA methylation level associated with heterosis of Epinephelus spp., we applied methylation-sensitive amplification polymorphism (MSAP) technology to detect the genomic DNA methylation levels of longtooth grouper (E. moara), giant grouper (E. lanceolatus), and their F1 hybrid [E. moara (♀) × E. lanceolatus (♂)], and we analyzed the difference of DNA methylation level between the F1 hybrid and parents. The results showed that overall genomic DNA methylation was higher in E. moara and E. lanceolatus. The total methylation rates of E. moara, E. lanceolatus, and the F1 hybrid were 60.62%, 59.38%, and 55.78%, respectively. The rates of fully methylated sites were 31.37%, 30.67%, and 29.27%, respectively, and the hemi-methylation rates were 29.25%, 28.71%, and 26.51%, respectively. The total methylation rate, the full methylation rate, and the hemi-methylation rate of DNA of the F1 hybrid were all significantly lower (P<0.01) than that of either parent. The full methylation rates were all higher than the hemi-methylation rates of E. moara, E. lanceolatus, and the F1 hybrid. The study also showed that the DNA methylation level of the F1 hybrid is negatively correlated with heterosis. Moreover, reduced DNA methylation level of the F1 hybrid may be one of the reasons for the heterosis phenomena such as rapid growth. In general, DNA methylation is negatively correlated with gene expression. The DNA methylation level of the hybrid was lower than either parent, allowing otherwise silenced genes to be activated, enhancing gene expression activity, and resulting in the hybrid progeny exhibiting heterosis. The relationship between methylation level of specific genes and heterosis, the genetic pattern of these DNA methylation sites, the effect on gene structure and function, and the regulatory mechanism require further study.

Abstract:The Chinese mitten crab Eriocheir sinensis is one of the most economically important aquaculture species in China due to its taste and nutritional value. In this study, the genetic status of both wild and cultivated stocks of E. sinensis was assessed using simple sequence repeat (SSR) markers. Ten microsatellite markers reported in previous studies were selected to estimate the level of genetic diversity within one Haihe natural population and three selected artificial populations (Yangtze No.1, Guanghe No.1, and Qilihai crab) and to compare the degree of genetic differentiation among them. Unique PCR products and high levels of polymorphism were observed for all loci. For the four populations of E. sinensis, the number of alleles per locus ranged from 3 to 17, with a mean allele number of 8.5 to 9.7. The mean observed heterozygosities ranged from 0.566 to 0.661, while the mean expected heterozygosities ranged from 0.720 to 0.745. Additionally, the mean polymorphism information content ranged from 0.687 to 0.716. Furthermore, the inbreeding coefficient (Fis) ranged from 0.080 to 0.827, and there were 13 cases that significantly deviated from the Hardy-Weinberg equilibrium (P<0.05) among the 40 population-locus cases (4 populations × 10 loci). The results of the microsatellite survey indicated that the genetic diversity of the three artificially selected populations was slightly lower than that of the natural population, but still maintained at a high level for further breeding. The analysis of genetic differentiation showed that Fst ranged from 0.015 to 0.075, genetic similarity ranged from 0.7702 to 0.9401, and genetic distance ranged from 0.0617 to 0.2611. UPGMA phylogenetic analysis divided these populations into two groups. Natural population and Guanghe No.1 stocks were allocated to the same cluster, and the Qilihai crab was allocated to another cluster. In conclusion, the four populations of E. sinensis possessed high genetic diversity but low to moderate levels of genetic differentiation. The information on the genetic variation and differentiation obtained in this study will provide a theoretical basis for further breeding and utilization of germplasm resources of E. sinensis.

Abstract:The Chinese mitten crab (Eriocheir sinensis) is an important freshwater crustacean cultured in China. It is popular with consumers because of its high nutritional value, pleasant smell, and taste. With increasing demand from the Chinese market, Chinese mitten crabs are now widely cultivated in ponds, lakes, and reservoirs. Pond culture of E. sinensis is the current main production method. To understand the parameters of morphological indicators of the Chinese mitten crab, some morphometrical traits, including carapace length (CL), carapace width (CW), carapace height (CH), and body weight (W) and moisture content were measured in E. sinensis cultured in ponds, and the hepatosomatic index (HSI) and muscle index (MI) were estimated to understand morphological attributes and quality parameters. The results showed no significant difference in CL, CW, body height, and MI in different sex groups of juvenile crabs (P>0.05), with significant differences in body weight in juvenile crabs (P<0.05). There were highly significant differences by sex in the above indices for adult crabs (P<0.01). Juvenile crabs had significantly higher HSI and MI than adult crab (P<0.01). The regression equation for juvenile crab body weight (Y1), body weight of adult crabs (Y2) CW (X2), and CH (X3) were expressed as Y1=12.067+16.416X3, and Y2=268.423+62.078X2, a higher decision coefficient for juveniles than for adults. The findings provide a scientific basis for ecological breeding of Chinese mitten crabs in yield estimation and quality evaluation.

Abstract:Ets transcription-factor networks represent a model for how combinatorial gene expression is achieved. The characteristic feature of Ets factors is the conserved ETS domain (Helix-Turn-Helix). Thus, the Ets proteins bind to a core GGAA/T consensus sequence and regulate expression of several genes and play an important role in various cellular functions (mitosis, growth, development, differentiation, and apoptosis) and the regulation of immunity. In this experiment, 9 Ets genes (named ETS 1~ETS9, respectively) of the Ark clam (Scapharca broughtonii) were successfully obtained by gene cloning technology, and the open reading frames (ORFs) were 1065 bp, 1290 bp, 1569 bp, 912 bp, 1344 bp, 1404 bp, 1521 bp, 1968 bp, and 1191 bp, respectively. Moreover, they encoded respectively 354, 429, 522, 303, 447, 468, 506, 655, and 396 amino acids. Evolutionary relationships of taxa showed that all genes in this chapter belonged to the Ets family genes. The qPCR detection showed that the expression of two Ets genes (ETS-1, ETS-3) was significantly increased. Thus, the present study showed that the Ark clam ETS-1 and ETS-3 are involved in the replication process of the OsHV-1 under high temperature conditions (16±2)℃. In conclusion, the results of this study provide a scientific basis for further study concerning the large number of deaths of Ark clams due to infection with OsHV-1 in summer.

Abstract:The metacaspase is a cysteine protease with the substrate of arginine/lysine, specifically found in protozoa, fungi, and plants. The metacaspase is generally believed to be closely related to the caspase of the metazoan. Previous studies have shown that metacaspases can be divided into type I and typeⅡ according to the differences in their structural characteristics, and both types have been found to be involved in the regulation and the control of programmed cell death in various plants and protozoa. Based on the information of the sequence of the Chlamydomonas reinhardtii type I metacaspase cDNA, obtained from the Genbank database (Genbank NO. XM_001696904), this study was able to use the method of two-step nested PCR cloning to retrive the Open Reading Frame (ORF) of the sequence of the C. reinhardtii type I metacaspase cDNA. The ORF sequence, mentioned above, was named as the CrMC1 in this study. The length of the CrMC1 ORF, in its entirety, is 987 bp, from which can be inferred that there are 405 amino acids encoded in this sequence. By the method of homologous sequence comparative analysis with the already known type I metacaspase protein, it was then found that the CrMC1 has conservative p20, p10, linker domain, and arginine and cysteine active center sites. Furthermore, this study is also able to show that, during the H2O2 induced programmed cell death of C. reinhardtii, the gene transcripts of the CrMC1 was increased at a statistically significant level (P<0.05). The gene transcripts of the CrMC1 then reached its peak after 2 h. Eventually, the gene transcripts of the CrMC1 decreased to the same level as it was in the control group at 3 h. In conclusion, the results as discussed above have indicated the following: the C. reinhardtii CrMC1 was involved in the regulation and the control of the H2O2 induced programmed cell death of C. reinhardtii.

Abstract:Marine macroalgae is used as a biofilter for aquaculture wastewater treatment. Marine macroalgae is used as a biofilter for aquaculture wastewater treatment. Ulva prolifera was cultivated at four different water temperatures (22.5℃, 25.5℃, 28.5℃, and 31.5℃) with a different inorganic nitrogen source (NH4Cl, NaNO2, and NaNO3) for each temperature to evaluate the purification efficiency of marine macroalgae U. prolifera on wastewater; all measurements were carried out in triplicate. The results show that, in the temperature range 22.5℃~31.5℃, the TAN uptake rates were 14.65, 14.88, 14.48, and 13.53 μmol/(g·h) in 96 h; the nitrite and nitrate uptake rates were 11.28, 10.48, 9.11, and 8.38 μmol/(g·h) and 9.41, 8.62, 8.80, and 7.35 μmol/(g·h) in 144 h, respectively. Both the temperature (P<0.01) and the nitrogen source (P<0.05) had significant effects on the growth rate of U. prolifera. The growth rate decreased as the temperature increased; further, at the same temperature with an ammonium source, U. prolifera showed the largest growth rate, followed by that with nitrite and nitrate sources. For the ammonium and nitrite sources, the content of chlorophyll a (Chl-a) increased with an increase in temperature; however, for the nitrate source, the content of Chl-a decreased first and then increased. Both the temperature and the nitrogen source had a significant effect on the carotenoid content (P<0.01), which increased with an increase in temperature. In the temperature range of 28.5℃~31.5℃, the carotenoid content in the nitrate source were found to be significantly higher than that in the other sources (P<0.05). The temperature and nitrogen source, thus, has a significant influence on the growth rate, Chl-a, and carotenoid content of U. prolifera. Overall, the TAN uptake rate was the highest, followed by the nitrite and nitrate uptake rates. However, the inorganic nitrogen uptake rate decreased as the temperature increased.

Abstract:Haematococcus pluvialis is a microalga, and this species is economically important since it is a rich source of natural astaxanthin, which is considered a “super anti-oxidant.” The study was conducted using H. pluvialis LUGU filtrated from Lugu Lake in Yunnan Province, China. Studies have shown that under stress, the exogenous addition of appropriate concentration of butylated hydroxytoluene (BHT) can effectively promote the accumulation of astaxanthin and increase the content of lipids in the algal cells. The effects of BHT on several traits were investigated under stress conditions (high illumination and nitrogen deficiency), including the impact on growth of algae; the accumulation of astaxanthin; the synthesis of lipids; the composition of fatty acids, carbohydrates and protein; the expression level of the key enzyme gene of astaxanthin; and fatty acid biosynthesis. In this study, the different concentrations of BHT (0, 1, 2, and 3 mg/L) were achieved through single factor experiments in algal cell culture medium. Results from these experiments showed that BHT treatment does not effectively promote the algae growth, but it does affect the accumulation of astaxanthin in algae cells. There was a significant dose effect depending on the BHT treatment applied to H. pluvialis. After examining BHT additions of different concentrations, astaxanthin accumulation was determined to be the highest after the 2 mg/L BHT treatment, and it was significantly higher than that resulting from other treatments or the control group (P<0.05). Moreover, astaxanthin accumulation was 1.87 times higher than that in the control group, reaching 31.66 mg/g. The lipid content of the 2 mg/L BHT treatment was 45.56%, which was also higher than that in the control (39.06%). Under these conditions, the expression levels of the key enzyme genes of astaxanthin synthesis, dxs and bkt, were 5.19 folds and 2.04 folds, respectively, as those of the control, and the expression levels of the key enzyme genes of fatty acid synthesis, kas and acp, were significantly higher than those of the control (P<0.05), being 4.56 folds and 3.02 folds, respectively, as those of the control. Contrastingly, the carbohydrate and protein contents decreased compared to those of the control group. Overall, our results also show that BHT can increase accumulation of astaxanthin and increase the content of lipids in the algal cells when administered at an appropriate dose.

Abstract:Forty-nine lactic acid bacteria strains were isolated from sediment samples from cultured ponds of sea cucumbers (Apostichopus japonicus) in Dongying, Shandong Province, in May 2017. First, antagonistic bacterial experiments were conducted using Vibrio splendidus and Pseudoalteromonas nigrifaciens, which are the main pathogenic bacteria of “skin ulcer syndrome” for sea cucumbers, and a lactic acid bacteria CLY-5, which has demonstrated significant bacteriostatic activity. Next, physiological and biochemical experiments and a similarity analysis of 16S rDNA sequences as well as the growth character of CLY-5 were carried out. A safety experiment for sea cucumbers was also performed. The results revealed that CLY-5 strain successfully inhibited V. splendidus and P. nigrifaciens. Meanwhile, the strain’s intracellular and extracellular products effectively inhibited V. splendidus and P. nigrifaciens, as demonstrated by inhibition zone diameters of 20 mm, 23 mm, and 27 mm, 38 mm, respectively, with extracellular products showing superior performance compared with intracellular products. Dipping bath concentrations were set to 1×109, 1×108, and 1×107 CFU/ml to test the safety of CLY-5 for sea cucumbers. During the experiment, all sea cucumbers were in good conditions. Based on the 16S rDNA sequence analysis, strain CLY-5 was determined to be identical to Lactobacillus plantarum NR117813.1. In addition, strain CLY-5 exhibited superior growth at 30℃~44℃ and salinities of 6 to 8. Moreover, CLY-5 came into a logarithmic growth phase after 20 h of cultivation and reached peak growth after 28~32 h. In summary, strain CLY-5 could be used to reduce the occurrence of sea cucumber disease and is also suitable for pond culture environment. These results provide a basis for ecological prevention strategies in disease control, and should prove useful in the development and utilization of lactic acid bacteria.

Abstract:In this study, Salmacis sphaeroides variegata was used as the research material to observe its morphology. Based on mitochondrial 16S rDNA gene partial sequencing, the genetic relationship between S. sphaeroides variegata and other echinoderms was analyzed. The nutritional composition of S. sphaeroides variegata gonad was analyzed to accumulate a mass of data for germplasm improvement and resource evaluation of S. sphaeroides variegata. The results showed that the main morphological characteristics of the S. sphaeroides variegata were as follows: the apical system was small, the outer edge of each step band plate had 3~4 small circular pits, and the bone fragment in the wall of the pedicle foot was C-shaped. The mitochondrial 16S rDNA gene sequences of other echinoderms and that obtained from S. sphaeroides variegata were compared by Mega 5.0, and the phylogenetic trees were constructed based on distance method (NJ) and maximum likelihood method (MP). The results showed that the S. sphaeroides variegata was closely related to the Salmacis bicolor, followed by the three species of sea urchins, the Temnopleuridae. An amount of water 86.1%, ash 3.0%, crude fat 2.0% and protein 8.01% was found in the sex glands of S. sphaeroides variegata. There were 16 kinds of amino acids in the sex glands of S. sphaeroides variegata, the total amino acid content was 38.71 g/100 g, EAA/TAA was 36.99%, EAA/NEAA was 71.84%, and DAA/TAA was 37.17%. A total of 15 fatty acids were detected, with the highest content being palmitic acid C16:0, stearic acid C18:0, EPA C20:5 and arachidonic acid C20:4, and the lowest content was that of C20:3. S. sphaeroides variegata showed a higher content of DHA than in other sea urchins. The results will provide basic data for the biological study of S. sphaeroides variegata and can be used for the development and application of sea urchin of economic value, as well as provide a theoretical basis for distant hybridization breeding and the germplasm identification of economically viable sea urchins.

Abstract:In this study, we obtained the full-length cDNA of the GHITM gene from Trionyx sinensis for the first time using the RACE (rapid-amplification of cDNA ends) method. The full-length cDNA sequence was 2650 bp, including a 123 bp 5-UTR, 1477 bp 3-UTR, and 1050 bp open reading frame (ORF) that encoded 349 amino acid residues. The isoelectric point (pI) of this peptide was 10.01, and the molecular mass was 37.12 kDa. The amino acid sequence was composed of the extracellular region, the transmembrane region, and the intracellular region. The transmembrane region was composed of 7 transmembrane domains. The phylogenetic analysis of the amino acid sequences showed that T. sinensis, Chrysemys picta bellii, and Chelonia mydas belonged to the same branch, the three species of crocodiles formed a branch, and birds formed another branch. The expression of GHITM in different tissues was also analyzed with quantitative real-time PCR. The results showed that GHITM was expressed in all the tested tissues, including the liver, pituitary, muscle, spleen, kidney, heart, intestine, and gonad, with the high expression observed in the liver, muscle, and pituitary. At the specifications of 50 g and 500 g, the expression of GHITM was significantly higher in the liver of males than females (P<0.05). Low temperature incubation can inhibit the expression of GHITM in fetal embryos (P<0.05). The results indicated that GHITM was related to the growth and embryo development of T. sinensis.

Abstract:Norovirus (NoVs) is the most prevalent worldwide foodborne pathogen and causes acute viral gastroenteritis. NoVs are transmitted mainly via the fecal–oral route and by person-to-person contact. It is thought that the majority of NoVs infections are caused by the consumption of contaminated food; the ingestion of contaminated oysters is the primary cause of foodborne NoVs infection since oyster digestive diverticula accumulate viral particles from seawater via filter feeding. Real time RT-PCR is commonly used to detect NoVs RNA in oysters; however, these assays are often hampered by the low viral titer in oysters and PCR inhibition due to matrix carryover during RNA extraction. Extraction is a critical step for obtaining sufficient high-quality viral RNA for amplification; however, studies comparing and evaluating NoVs RNA extraction and detection methods in shellfish are often limited by the lack of standard samples with clear quantitative values and a lack of biosafety hazards. In this study, four RNA extraction methods (TRIzol reagent, Viral RNA Kit, High Pure Viral Nucleic Acid Kit, and Column Virus RNAOUT Kit) were used on oyster digestive gland homogenate samples and artificial freeze-dried samples contaminated with NoVs armored RNA (3.01010 copies/sample) as a reference material. RNA extracted by the four methods was analyzed by real time RT-PCR and quantified using previously established standard curves. For the homogenized samples, the TRIzol method had the highest recovery rate (6.8± 0.89)% and was significantly higher than that by the Viral RNA Kit (4.51±2.28)%. The recovery rates of these two methods were both significantly higher than those by the High Pure Viral Nucleic Acid Kit (0.24±0.05)% and Column Virus RNAOUT kit [(0.11±0.02)%, P˂0.05]. For the freeze-dried samples, the Viral RNA Kit had the highest recovery rate (8.71±0.17)% and was significantly higher than that by the TRIzol method (7.12±0.64)%. The recovery rates of these two methods were both significantly higher than those of the High Pure Viral Nucleic Acid Kit (0.33±0.12)% and Column Virus RNAOUT kit [(0.06± 0.01)%, P˂0.05]. This study indicated that the TRIzol method and Viral RNA Kit could extract target RNA from oyster digestive gland homogenate samples with an ideal recovery rate; moreover, armored RNA could serve as a good reference material for comparing RNA extraction methods.

Abstract:Turbot is internationally recognized as a high-value edible fish and has become one of the important farmed fish in the northern coast of China; however, its nutritional quality still lacks comprehensive evaluation, and its deep processing rate is too low. To provide fundamental data for the segmentation processing of turbot, the nutritional components, content of collagen, amino acid, fatty acid, and texture characteristics of different parts of turbot, including the dorsum, abdomen, chest, tail, skirt, and skin, were determined. Meanwhile, the nutritional value was also evaluated. The results showed that there were differences in crude protein content from different parts, among which crude protein content of skin was the highest (29.04%) and that of skirt was the lowest (12.99%), both of which were significantly different from that of the other parts (P<0.01): the crude protein content from the dorsum, abdomen, chest and tail was 18.76%, 18.96%, 17.91%, and 18.39%, respectively. The maximum fat content in the skirt was as high as 17.47%, containing the most types of fatty acids (27 kinds), and reached 318.09 mg/g; Polyunsaturated fatty acid (PUFA) was 3.90 to 6.76 times as much as that of the other parts, among which linoleic acid content was the highest (107.26 mg/g), followed by docosahexaenoic acid (DHA) (64.39 mg/g) and eicosapentaenoic acid (EPA) (26.61 mg/g). The skin had the highest level of 224.69 mg/g in collagen (P<0.01), which could be used as a raw material to produce collagen. A total of 18 amino acids were detected in all parts of the turbot. The main limiting amino acids in the dorsum, abdomen, chest, tail, and skirt were all methionine (Met) and cysteine (Cys), while the main limiting amino acid in the skin was tryptophan (Trp). In addition, except for the skin, amino acid compositions in other parts of the turbot were all in line with the ideal pattern described by FAO/WHO. Texture characteristics analysis showed that hardness, chewiness, and elasticity of the abdomen were significantly higher than those of the other parts (P<0.05), which had a better mouthfeel.

Abstract:The hairtail (Trichiurus haumela) is rich in protein and high in nutrition. In this study, the effects of chilling storage on the muscle protein of hairtail were studied by measuring a series of physical and chemical indicators that characterize changes in the fish quality, including TCA-soluble peptides, sulfhydryl group content, surface hydrophobicity, Ca2+-ATPase activity, and Mg2+-ATPase activity, as well as SDS-PAGE patterns of total soluble proteins, water-soluble proteins, low-salt-soluble proteins, and high-salt-soluble proteins. The effects of 0℃ and 4℃ chilling on the fish muscle protein were observed after 15 days of storage, and the oxidation, denaturation, and degradation of the fish protein under the two storage temperatures were compared. The results showed that after 0℃ and 4℃ storage for 15 days, the TCA-soluble peptides increased from 1.446 μmol/g to 6.717 μmol/g and 7.595 μmol/g, respectively. The Ca2+-ATPase activities were lower than the initial values, decreasing from 0.99 U/mg prot to 0.092 U/mg prot and 0.134 U/mg prot, respectively. The Mg2+-ATPase activities also decreased from 0.76 U/mg prot to 0.199 U/mg prot and 0.125 U/mg prot, respectively. In addition, the sulfhydryl group content decreased from 38.15 μmol/g MP to 35.82 μmol/g MP and 30.36 μmol/g MP, respectively. The surface hydrophobicity first increased and then decreased, but the values were higher than those of the fresh samples. The changes in the values of this series of physical and chemical indicators indicated that the refrigerating conditions of 0℃ and 4℃ were not conducive to the long-term storage of the hairtail. Prolonged storage time destroyed the freshness and quality of the hairtail owing to protein oxidation, which caused a bad odor and rendered the fish inedible. From the microstructure perspective, there were almost no changes in the total soluble proteins and water-soluble proteins. However, the high-salt-soluble proteins degraded under storage, indicating that the myofibrillar protein had degraded gradually, decreasing the fish quality. Moreover, the rate of myofibrillar protein degradation and corruption of the fish was slower at 0℃ than at 4℃ on the same day during storage.

Abstract:In this study, chitosan-citrus essential oil microcapsules were prepared with the following three ingredients at varying concentrations: citrus essential oil 6 g/L, emulsifier 3 ml/L, TPP(Thiamine pyrohphosphate) 2 g/L, and chitosan at 2.5, 5.0, 7.5, 10, 15, or 20 g/L; citrus essential oil 6 g/L, TPP 2 g/L, chitosan 10 g/L, and emulsifier at 0, 3, 6, 9, or 12 ml/L; and citrus essential oil 6 g/L, emulsifier 3 ml/L, chitosan 10 g/L, and TPP at 0.5, 1, 2, 4, or 8 g/L. The particle size, PDI(Poly-dispersible), and zeta potential were used as indicators, and chitosan 10 g/L, citrus essential oil 6 g/L, emulsifier 9 ml/L, and TPP 4 g/L were eventually selected as the final ingredient concentrations for preparing the microcapsules. After soaking Litopenaeus vannamei with 1% (w/v) microcapsule solution for 20 min, the total volatile basic nitrogen (TVB-N) content, SDS-PAGE patterns, thiobarbituric acid (TBA) content, and total viable count (TVC) were analyzed to determine the effectiveness of the chitosan-citrus essential oil microcapsules in preserving the quality of L. vannamei during storage at 4℃. The results showed that the TVB-N level of the experimental group increased slowly (P<0.05), reaching 27.47 mg/100 g on the 22nd day of storage, which was at the secondary freshness level, whereas that of the control group exceeded the secondary freshness level on the 19th day of storage. Likewise, the TBA level of the experimental group increased slowly (P<0.05), reaching 0.789 mg/kg on the 22nd day of storage, which was the same level as that of the control group on the 15th day. The TVC of the experimental group also increased slowly (P<0.05), reaching 5.42 [lg (CFU/g)] on the 19th day of storage, which was at the secondary freshness level, whereas that of the control group exceeded the secondary freshness level on the 13th day of storage. The SDS-PAGE patterns showed that the myosin heavy chain of the experimental group had decomposed more slowly. These results indicated that the chitosan-citrus essential oil microcapsules could effectively inhibit the degeneration of protein, the rancidity of fat, and the growth of microorganisms, thus, hindering the spoilage of L. vannamei and extending its shelf life by 3~4 days compared with that of the control.