Abstract:In recent years, global warming and the release of hot wastewater from nuclear power plants have caused the temperature of sea water to rise, leading to severe thermal pollution of the oceans. Marine ecosystems and the survival of marine life face huge challenges from ocean warming. Research on the thermal tolerance of marine organisms will help us to understand the heat tolerance of different marine species and determine how to control thermal discharge from nuclear power plants. This study investigated the heat tolerance of four marine species (Chaeturichthys stigmatias, Sebastes schlegeli, Oratosquilla oratoria, and Charybdis japonica) in Liaodong Bay using a dynamic temperature-rising test. The lethal temperature of 50% (LT50) was assessed at five acclimation temperatures (8.0℃, 14.0℃, 18.0℃, 24.0℃, 28.0℃) and nine temperature-rising rates (0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 9.0, 12.0, 15.0℃/h) to evaluate heat tolerance. The results showed that both the acclimation temperature and temperature-rising rate significantly affected the LT50 value of the experimental animals (P<0.05). The LT50 values were significantly positively correlated with the acclimation temperature (P<0.05), while the effects of the temperature-rising rate varied with acclimation temperature in a species-dependent manner. In this study, the dynamic temperature-rising test included a higher number of acclimation temperatures and temperature-rising rates than used in previous studies, and we established the LT50 value as an important indicator for evaluating the heat tolerance of marine species. These results suggest that as an evaluated index, LT50 comprehensively reflects the heat tolerance of experimental species, which is of scientific and ecological significance and could be widely used in the assessment of heat tolerance.

Abstract:Based on phytoplankton net samples from the Bohai Sea from 1959 to 2015, the long-term changes in species composition, cell abundance, community structure, and species diversity were analyzed. A total of 77 genera and 170 taxa were recorded, most of which were temperate-coastal ecotype species. The composition pattern of the dominant taxa changed dramatically over the fifty-plus years. Centric diatoms such as Chaetoceros spp. and Coscinodiscus spp. dominated in the communities of last century, while Paralia sulcata, Thalassionema spp., and dinoflagellates such as Noctiluca scintillans and Tripos spp. gradually became predominant in this century. The cruise-averaged cell abundances ranged from 8.33 × 104 cells/m3 to 472 × 104 cells/m3, with diatoms accounting for 65.3%~99.8%. The abundances and species diversities declined to their lowest levels last century, whilst they had 1.5 times and 15.0% respective recoveries in this century. The persistent increase in seawater N∶P ratios in the Bohai Sea has led to a phytoplankton transition from diatom-dominated communities to communities co-dominated by diatoms and dinoflagellates, with an increase of 2.82 times in the ratio of diatoms to dinoflagellates in this century from that in the last century. The decadal changes in the phytoplankton community structure and its herbal diet basis in the Bohai Sea affect the recruitment process during the early life stages of the key resource species. This study provides a baseline database and references for discussion on the mechanisms of adaptive responses of fishery populations to the long-term environmental changes in the Bohai Sea.

Abstract:In this study, data from surveys of fishery resources and environments collected from the Bohai Sea ware used to construct an Ecopath model of 1982 and 2014~2015, respectively. Based on the models, we analyzed the trophic impact relationship, structure, and functional parameters of the Bohai Sea ecosystem. Furthermore, we calculated the ecological carrying capacity for Chinese shrimp in the Bohai Sea and compared its changes between the two periods. Trophic impact and keystoness index results showed that the Chinese shrimp was not a keystone species or an important functional group of this ecosystem. Benthic crustaceans, mollusks, and other functional groups occupied important trophic positions in the Bohai Sea ecosystem. The increase in Chinese shrimp biomass will have a negative impact on Oratosquilla oratoria, Portunus trituberculatus, other benthic crustaceans, and polychaetes. The increase in the biomass of functional groups such as Lateolabrax japonicus and gobies may have a negative impact on Chinese shrimp. Network analysis showed that this ecosystem was at an unstable developmental stage during these two periods, and the Bohai Sea ecosystem had higher surplus production. A comparison of the 1982 and 2014~2015 models showed that the maturity and stability of the ecosystem decreased, resulting from its degeneration. The carrying capacity of Chinese shrimp was found to be 0.810 and 0.702 t/km2 in Bohai Sea in 1982 and 2014~2015, respectively. Compared with its current biomass, Chinese shrimp had great potential for continuous enhancement, e.g., by releasing larvae in the Bohai Sea, and the ecological carrying capacity was not exceeded even when the biomass of the Chinese shrimp was increased by 71.68 and 585 fold in 1982 and 2014~2015, respectively. This study provides information for rational implementation of stock enhancement and promotion of sustainable development of fisheries resources in this sea area.

Abstract:Genome editing in fish has many potential applications in aquaculture and is important for gene functional analyses and genetic engineering breeding. Furthermore, genome editing in aquaculture animals is useful for cultivating new varieties and researching disease resistance and aquaculture growth. However, the difficulty of microinjection and cultivation in mariculture fish embryos seriously hampers the application of genome editing technology. Many methods are used to introduce DNA, RNA, and protein to fish genomes, such as pulsed electric, retroviral transfection, and laser-mediated technologies. Microinjection is the most widely used method. Turbot (Scophthalmus maximus) is one of the most important mariculture fish in Chinese aquaculture. To our knowledge, there have been no reports on the success of the microinjection technique in turbots. In this study, the smyd1 : gfp plasmid (50 ng/µl) with green fluorescent protein (GFP) expression by a 5.3 kb muscle-specific smyd1 promoter fragment was successfully microinjected and expressed in the cytoplasm of newly zygote of turbot at the 1~4 cell stage. The effects of microinjection on the survival rate of turbot embryos were studied. The survival rate of microinjected embryos was lower than that of uninjected embryos. Overall, 120 microinjected fishes (4.8%) survived and 190 (19.0%) uninjected survived. The GFP expression in the embryos and larvae was observed using a fluorescence microscope (Nikon Eclipse 80i). In total, 56% (28/50) of microinjected fishes expressed GFP. A specific GFP-encoding segment (346 bp) was amplified from the DNA of microinjected embryos and larvae by polymerase chain reaction, showing that the GFP gene had been introduced into the turbot embryos and confirming successful microinjection technique of turbot.

Abstract:This study aimed to investigate the transcriptional regulation of a teleost antimicrobial peptide. The expression pattern of the rainbow trout Cathelicidin2 (Cath2) gene was analyzed by quantitative real-time PCR (qRT-PCR). Cath2 was expressed in tissues closely related to immune defense, including the gill and head kidney, and expression significantly increased after both bacterial and viral infections. Promoter and transcription factor binding sites were analyzed for the upstream regulatory sequence of Cath2 gene. The predicted promoter contained characteristic eukaryotic TATA and CAAT boxes, as well as multiple binding sites for immune-related transcription factors, including two candidate binding sites for nuclear factor kappa B (NFκB) on the positive strand of the core promoter. In Ctenopharyngodon idella kidney cell lines, transcription of both green fluorescent protein and firefly luciferase genes was initiated by the predicted Cath2 promoter. Both the bacterial mimetic lipopolysaccharide and the viral mimetic polyinosinic polycytidylic acid up-regulated promoter activity. As shown in the dual luciferase reporter assay, promoter activity was enhanced by co-expression of the transcription factor NFκB, indicating that Cath2 is regulated by the NFκB pathway. These results suggest that expression of the rainbow trout Cath2 gene could be induced by different immune stimuli, and its promoter might serve as an immune-inducible transgenic element. The Cath2 promoter may initiate the transcription of heterologous immune genes against exogenous infection in a proper expression pattern, avoiding excessive transcription under unnecessary conditions; therefore, it has potential for genetic engineering approaches in the breeding of disease-resistant fish.

Abstract:DNA methylation plays an important role in regulating gene expression, cell aging and trait differentiation. In order to investigate the differences of DNA methylation level and pattern in different genders and tissues of Sepia esculenta Hoyle, we applied Fluorescence-labeled Methylation Sensitive Amplified Polymorphism (F-MSAP) technology and analyzed the level and pattern of methylation of genomic DNA in the muscle, the heart, the pancreas and the gonad of female and male S. esculenta. The total methylation rate of S. esculenta in the growing stage was 23.97%~39.70%, which was at a high level in aquatic invertebrates. Among the female and male's four tissues, the total methylation level in muscle was the highest, which may be related to the phenomenon of allometric growth and the preferential development of the muscle during the growing state. Moreover, both the methylation levels and patterns of S. esculenta were different in gender, the total methylation level of DNA in the muscle tissue of the female S. esculenta was significantly lower than that of the male, but that in the heart and pancreas tissue was significantly higher than that in the male. In addition, we also found that the fully methylation level in the muscle of female S. esculenta was the largest contributor to the total methylation level and was consistent with the level of total methylation in the other tissues of female S. esculenta and in the four tissues of male S. esculenta. The results showed that the level and pattern of DNA methylation of S. esculenta had gender and tissue differences. This study can provide basic data for epigenetic regulation of the life processes such as the growth, differentiation, and death of S. esculenta.

Abstract:Four isonitrogenous, isoenergetic, and isolipidic diets with varying ratios (0 : 6, 2 : 4, 4 : 2, and 6 : 0) of concentrated soybean phospholipid oil (P) to refined rapeseed oil (R) (P0R6, P2R4, P4R2, and P6R0) were formulated to determine the suitable ratio of soybean phospholipid oil to rapeseed oil in the diet of juvenile large yellow croaker (Larimichthys crocea). Each diet was randomly assigned to triplicate sea floating cages of 40 fish [initial body weight (20.84±0.05) g]. Fish were fed to apparent satiation twice daily (5:30 and 16:00) over 51 d. The results suggested that the survival rates of tested fish were higher than 91%, with no significant differences among treatments (P>0.05). The special growth rate was significantly higher with fish fed P2R4, compared with those fed P4R2 and P6R0 (P<0.05). Higher lipid and protein deposition rates were found in P2R4-fed fish compared with those fed the other treatments (P<0.05). Contents of crude protein in whole body and muscle were not significantly affected by the ratio of soybean phospholipid oil to rapeseed oil in diets (P>0.05), but were significantly higher in the viscera of P0R6-fed fish compared with the other treatments (P<0.05). Concentrations of crude lipid in the whole body of P0R6- and P2R4-fed fish were significantly higher than those in P4R2-and P6R0-fed fish (P<0.05). The fatty acid profile of the whole body and muscle was similar to that of the fed diet. The ratio of soybean phospholipid oil to rapeseed oil in diets had no significant effect on hepatosomatic index and viscerosomatic index (P>0.05). The P6R0 diet significantly reduced the liver lipid content and condition factor of test fish (P<0.05). Under the conditions of this study, the suitable ratio of concentrated soybean phospholipid oil to refined rapeseed oil in the diets of juvenile large yellow croaker was 2 : 4, based on growth performance, body composition, and fatty acid composition.

Abstract:An 8-week feeding trial was conducted in a recirculated system to study the effect of dietary n-3/n-6 long-chain polyunsaturated fatty acid (LC-PUFA) ratios (29.54, D1; 23.04, D2; 18.97, D3; 9.06, D4; 6.86, D5; 3.87, D6) on growth performance, whole-body fatty acid composition, and serum biochemical indices in turbot (Scophthalmus maximus). Six diets were formulated to feed six groups of juvenile turbots [mean initial weight (12.18±0.01) g]. Each diet was randomly fed to triplicate groups of 35 fish per tank. There were no significant differences in the survival rate (SR) of turbot (P>0.05). With decreasing dietary n-3/n-6 LC-PUFA ratios, the weight gain rate (WGR) first increased and then decreased. The WGR of the D6 group was significantly lower than that of the other groups (P<0.05). The lipid deposition rate (LR) reduced with decreasing dietary n-3/n-6 fatty acid ratios, and the D6 group achieved 14.80, which was significant lower than that of the other group (P<0.05). The trypsin content increased as the dietary n-3/n-6 LC-PUFA level decreased from 29.54 to 9.06, and then decreased. Lipase increased with decreasing n-3/n-6 LC-PUFA. As the dietary n-3/n-6LC-PUFA level decreased, fatty acid synthetase and glucose-6-phosphate dehydrogenase first increased and then decreased. When the dietary n-3/n-6 LC- PUFA level was 9.06, fatty acid synthetase levels were significantly higher than those in the other groups (P<0.05). Additionally, glucose-6-phosphate dehydrogenase levels were significantly higher than those in the other groups (P<0.05) when the dietary n-3/n-6 LC-PUFA level was 18.97. Aspartate aminotransferase, total protein, albumin, and high-density lipoprotein cholesterol first increased and then decreased with increasing dietary n-3/n-6 LC-PUFA ratios. These results indicate that, with decreasing dietary n-3/n-6 LC-PUFA ratios, resulting in reduced lipid deposition rate of juvenile turbot.

Abstract:A study was conducted to investigate the effect of feeding frequency on growth, reproductive performance, body composition and digestive enzyme of broodstock Litopenaeus vannamei. Experimental broodstock were fed with Nereis succinea for 60 days. Before the start of the experiment, the unilateral eye socket of each broodstock was placed on the eye as an individual marker and the initial body length and weight of each female were determined. The average initial body weight was 41.78 g and the average initial body length was 15.80 cm. The broodstock were randomly divided into three round pools, each with 40 shrimps. Three groups of broodstock were fed to satiation in two times per day, three times per day and four times per day, which were represented by F2, F3 and F4 groups, respectively. Results showed that the feeding amount of broodstock increased gradually with the increase of feeding frequency, and the F2 group was significantly lower than that in group F4 (P<0.05), but there was no significant difference between the F3 group and the F2 group (P>0.05). The weight gain and body length growth of broodstock increased first and then decreased with the increase of feeding frequency, but there was no significant differences among treatments (P>0.05). Reproductive performances of Litopenaeus vannameimei were not affected by different feeding frequency in current study (P>0.05). There were no significant differences in single fecundity, relative fecundity, the number of nauplius stage I, hatching rate, the number of zoea stage metamorphosis rate (N1-Z1), gonadosomatic index(GSI),first spawning time and the spawning intervals (P>0.05). The body composition of Litopenaeus vannamei including moisure, crude protein, crude Lipid and ash were not affected by feeding frequency (P>0.05). The results showed that the activity of trypsin and lipase of hepatopancreas in the F2 group was significantly lower than that in the F3 and F4 groups (P<0.05), but there was no significant differences among the activity of pepsin and amylase in different experimental groups (P>0.05). Based on the results of this experiment and the actual production conditions, the optimum feeding frequency of nutrient fortification in Litopenaeus vannamei broodstock is suggested to be 2 times per day.

Abstract:Growth-related parameters of individuals from four populations of Litopenaeus vannamei from Huanghua of Heibei Province (HH), Pingdu of Shandong Province (PD), Wujiang of Jiangsu Province (WJ), and Rizhao of Shandong Province (RZ) were assessed. Amount of Enterocytozoon hepatopenaei (EHP) in the hepatopancreas of individuals from all populations and EHP in multiple tissues of shrimp from the RZ population were assessed with TaqMan-based quantitative PCR. The results showed that the RZ population possessed optimal growth-related parameters and the lowest EHP among the four populations. The histograms of case-logarithmic EHPs of the four populations presented different modes. The case distribution of logarithmic EHPs from the HH and PD populations showed double peaks, while those of the WJ and RZ populations showed a single peak. The different distribution modes may indicate a different EHP spread in the four populations. The population with a single peak mode or the higher logarithmic EHP subpopulation isolated from the population with a multiple peak mode showed a significant negative correlation with shrimp body length or body weight to logarithmic EHP. The EHP detected in different tissues of the RZ population followed the order (from highest to lowest), EHP in hepatopancreas > EHP in midgut > EHP in hemolymph > EHP in gills > EHP in muscle. The logarithmic EHP in the hepatopancreas–midgut, hepatopancreas–gills, and midgut–gills had a significant correlation level above 99.9% (P<0.001), while the logarithmic EHP of the other two tissues had a significant correlation level above 99.0% (P<0.01) or above 99.5% (P<0.05), except for midgut–hemolymph and hepatopancreas–hemolymph. In-situ hybridization of a DIG-labeled EHP probe in the hepatopancreas, muscle, gills, and midgut showed that the hepatopancreas is the major target tissue of EHP infection in shrimp. Minor and weak hybridization signals were also observed in other tissues, which indicated that a few cells in those tissues were also susceptible to EHP infection in L. vannamei.

Abstract:Acute hepatopancreatic necrosis disease (AHPND) results from acute toxicity in the hepatopancreas of infected shrimp caused by the toxic proteins PirAVp and PirBVp, which are expressed by the pVA1 plasmid carried by AHPND-causing Vibrio parahaemolyticus (VpAHPND). In this study, Litopenaeus vannamei were exposed to 2.19×105 CFU/ml VpAHPND strain 20130629002S01 by immersion to explore the dynamic changes of VpAHPND in the tissues of shrimp. The hepatopancreas, gills, midgut, and muscle of infected shrimp were collected 2~9 days after immersion infection, and the quantity of pirAVP was measured by qPCR. The results showed that VpAHPND could be detected in all sampled tissues of infected shrimp. The amount of VpAHPND in the hepatopancreas reached a peak on day 4 post-infection at 8.71×104 copies/mg, while the gills, muscle, and midgut reached peaks on day 3, 4, and 5 post-infection at 9.08×103、2.59×104、5.76×104 copies/mg, respectively. The highest amount of VpAHPND was detected in the gills during the early stage of infection, followed by the hepatopancreas and midgut in sequence during heavy disease, with frequent deaths. Subsequently, the amount of VpAHPND declined rapidly in all tissues, with similar levels in the midgut, hepatopancreas, and muscle. Histopathology revealed that AHPND lesions were denser in hepatopancreas samples from moribund shrimp compared with those from morbid shrimp, when taken at the same time from infection. Furthermore, the histopathologic symptoms of both became more severe along the infection process, but with decreasing levels of VpAHPND. The results showed that the copy number of pirAVp in tissues of VpAHPND-infected shrimp does not represent the real-time condition of diseased shrimp and the quantity of VpAHPND may not be high in a severe AHPND sample.

Abstract:In order to understand the regulatory mechanism of ovarian development in Exopalaemon carinicauda, we used RACE (Rapid amplification of cDNA ends) technology to clone the translationally controlled tumor protein (TCTP) gene. The full-length cDNA of TCTP gene in E. carinicauda (EcTCTP) is 732 bp long with an intact open reading frame of 507 bp encoding a polypeptide of 168 amino acids. Six kinds of functional sites were identified in EcTCTP, including one N-glycosylation site, three protein kinase C phosphorylation sites, six casein kinase Ⅱ phosphorylation sites, three N-myristoylation sites, TCTP domain signature 1, and TCTP domain signature 2. Multiple alignment analysis and phylogenetic tree revealed that EcTCTP has the highest similarity to TCTP from Eriocheir sinensis. The results demonstrated that the cysteine (Cys) residue at the C terminal is highly conserved in all the animals, except the crustaceans. The lack of Cys residue might affect the oxidation resistance and the formation of TCTP dimers in crustaceans. The expression profile of 4 kinds of autophagy-related genes during the ovarian development period showed that: (1) EcTCTP gene in the hepatopancreas from proliferative stage to postpartum recovery period decreased. (2) Expression of EcTCTP gene in the ovary was the highest during the minor growth phase. Furthermore, the expression profile of Hif-1α, Beclin1, and Bcl-2 in hepatopancreas showed a similar trend, that is, high expression from proliferative period to minor growth phase, the lowest expression during the major growth phase with an increase during the mature period. The expression level of the gene Hif-1 in the ovary was similar to that of the gene vitellogenin in the ovary, which increased from the proliferative phase to the postpartum recovery period. The expression level of the gene Beclin1 in the ovarian cells increased from the proliferative stage to the major growth phase, and reached the highest level during the major growth phase, which was consistent with the synthesis of endogenous vitellogenin during the late development stage in prawn. The expression of the gene Bcl-2 in the ovary was the lowest during the major growth phase. The results illustrated that the autophagy-related genes might function together in the ovarian development of E. carinicauda by regulating autophagy during the synthesis of vitellogenin.

Abstract:Based on the cloning of Exopalaemon carinicauda E75 (EcE75, GenBank accession number: KY471317), we studied its role in molting and the expression pattern of EcECR and EcRXR during different stages of molting. The full-length cDNA of EcE75 is 3944 bp long, which contains an open reading frame (ORF) of 2520 bp. The protein encoded by EcE75 is composed of 839 amino acids. The estimated mass of the deduced amino acid sequence of EcE75 is 92.307 kDa, and the calculated theoretical isoelectric point (pI) is 7.48. The ZnF_C4 and the HOLI domains were detected in EcE75, and one distinct transmembrane helix was identified. The gene EcE75 is conserved during evolution. The phylogenetic tree constructed based on E75 sequences showed that EcE75 was clustered with that of the same clade of crustaceans. E. carinicauda has the highest homology with Fenneropenaeus chinensis, Portunus trituberculatus, Litopenaeus vannamei, and Gecarcinus lateralis. The tissue distribution analysis showed that EcE75 transcript could be detected in all tested tissues of E. carinicauda, and relatively abundant in eyestalk and ovary. The expression pattern of the genes EcRXR, EcECR, and EcE75 was consistent during different stages of molting. At the premoult and postmoult stages, the expression of genes of reproductive molting was significantly higher than that of growth molting. The results showed that there was significant difference between the growth and reproductive molting because of the ovary development. Furthermore, it revealed the effect of ecdysone stimulation on ovarian development through the upregulation of expression of the genes EcRXR, EcECR, and EcE75. The present study on the expression of the genes EcRXR, EcECR, and EcE75 at different stages of molting provides a theoretical basis and a scientific ground for further research on the mechanism of molting.

Abstract:Arcuatula senhousia is a high-quality food for shrimps, crabs, and other aquaculture species and is a type of shellfish with a high breed latent capacity. The effects of temperature (7℃, 15℃, 23℃, 31℃) on the physiological metabolism of A. senhousia were studied using the stagnant water method under controlled laboratory conditions in October 2015, to obtain a deeper understanding of its physiological metabolism and to establish its energy budget. The results showed that the filtration rate, feeding rate, oxygen consumption rate, and assimilation efficiency of A. senhousia at 23℃ were significantly higher than those at other temperatures (P<0.05), by up to 1.09 L/(g·h), 24.46 mg/(g·h), 3.50 mg/(g·h), and 62.93%, respectively. The filtration rate, feeding rate, oxygen consumption rate, fecal pellet production rate, and ammonia excretion rate of A. senhousia at 7℃ were significantly lower than those at other temperatures (P<0.05), as low as 0.24 L/(g·h), 6.04 mg/(g·h), 1.02 mg/(g·h), 4.20 mg/(g·h), and 2.33 μmol/(g·h), respectively. With increased temperature, the filtration rate, feeding rate, oxygen consumption rate, and assimilation efficiency of A. senhousia increased and then decreased, reaching the highest value at 23℃. The energy budget equations at different temperatures are as follows: 100C=58.12F+46.74R+2.54U–7.40P (7℃); 100C=44.28F+29.14R+1.85U+24.73P (15℃); 100C=17.18F+41.81R+6.64U+34.37P (23℃); 100C= 53.35F+28.26R+14.66U+3.73P (31℃); The energy budget equations show that the energy required for growth (P), respiration (R), excretion (U), and feces (F) accounts for –7.4% to 34.37%, 28.26% to 46.74%, 1.85% to 14.66%, and 17.18% to 58.12% of the feeding energy (C), respectively. The proportion of growth energy to feeding energy varies greatly at different temperatures. Growth energy accounts for the highest percentage (34.37%) at 23℃ (P<0.05) and the lowest percentage (–7.40%) at 7℃. The results of this study provide theoretical guidance for a deeper understanding of the physiological energetics of A. senhousia.

Abstract:Onchidium struma belongs to Mollusca, Pulmonata, and was regarded as the transitional taxa from water to land because of its amphibious characteristics. Having similar characters to amphibians, O. struma is a good subject for the study of marine invertebrates extending landward. As the basic component of muscle units, myosin directly influences the muscle growth and meat quality via its molecular diversity and composition. It is a ubiquitous eukaryotic motor protein that interacts with actin to generate the force for cellular movements, ranging from cytokinesis to muscle contraction. In this study, we used rapid amplification cDNA ends (RACE) methods to obtain the full-length cDNA of the myosin heavy chain (MyHC) gene in O. struma. We performed bioinformatic and expression pattern analysis of MyHC mRNA in different tissues detected by real-time fluorescent quantitative PCR (qRT-PCR). The full length of the MyHC cDNA sequence consists of 7566 base pairs (bp) comprising a 228 bp 5' untranslated region (UTR), a 1443 bp 3' UTR, and a 5895 bp open reading frame (ORF) which encodes 1964 amino acids. MyHC gene was expressed in various tissues; its highest expression was found in the foot and its lowest in the hepatopancreas (P<0.05). Moreover, the MSTN protein was predicted to be composed of 31,713 atoms and its formula is C9765H15897N2849O3150S52, with a calculated relative molecular weight of 225.28 kDa and a pI of 5.56. The result of signal peptide prediction shows that the N-terminal has a signal peptide of 29 amino acids in length. Additionally, common features were found in the MyHC of O. struma, including MYSc class II and myosin tail l domain. Molecular phylogenetic analysis shows that O. struma is closely related to Biomphalaria glabrata. This study provided a novel myosin heavy chain gene sequence in O. struma and the results indicate that the MyHC gene is important for the growth and development of this animal, as well as its muscle characterization. Furthermore, the results revealed that MyHC is not only an essential structural protein, but also a functional protein in O. struma. Conclusively, MyHC is an excellent candidate gene for studying biological evolution.

Abstract:In this study, expressed sequence tag (EST)-SSR markers were developed to investigate the genetic relationship between two commercially important scallop species, Yesso scallop Patinopecten yessoensis and Zhikong scallop Chlamys farreri. A total of 60 EST-SSRs previously developed from P. yessoensis were selected, and their cross-species amplification in C. farreri was analyzed. As a result, 21 pairs of EST-SSR primers showed unique PCR products. The interspecies transferability was calculated to be 35.00%. Among the 21 EST-SSR primers, 17 were polymorphic in the studied populations, which resulted in 28.33% transferability. In the two populations of P. yessoensis and C. farreri, the number of alleles ranged from 2.00 to 4.00, with mean allele numbers of 2.7647 and 2.3529, respectively. The mean effective number of alleles was estimated to be 1.9487 and 1.6350, while the mean observed heterozygosity was 0.6314 and 0.3333, respectively. Similar to the observed heterozygosity, the mean expected heterozygosity was higher in P. yessoensis (0.4569) than in C. farreri (0.3139). For the two populations, the diversity index was consistently higher in P. yessoensis than in C. farreri. The mean polymorphism information content was estimated to be 0.3726 and 0.2597, and Nei’s (1973) gene diversity index was calculated to be 0.4493 and 0.3087, respectively. Furthermore, Shannon’s information index in the two populations was 0.7176 and 0.5041, respectively. The genetic identity between the two species was calculated to be 0.619, with a high genetic divergence between them (0.480). Among the polymorphic markers, seven loci significantly deviated from the Hardy-Weinberg equilibrium according to the average fixation index (Fis). The genetic differentiation index (Fst) between the two populations was estimated to be 0.2398. The EST-SSR markers developed for cross-species amplification of P. yessoensis and C. farreri are important resources for the study of genetic diversity, marker-assisted breeding, gene discovery, and genetic evaluation of germplasm resources.

Abstract:Scapharca broughtonii (Mollusca, Bivalve, Arcoida), one of the most important commercial marine bivalve species, is mainly distributed on the coasts of the Bohai Sea, and north of the Yellow Sea, China. With the deterioration of marine ecological environments resulting from the extended farming scale and frequency of human activities in coastal waters, and over-fishing, wild resources have decreased. Mass mortality has become a major constraint for the development of S. broughtonii culture. The hybridization of different populations has proven to be a good breeding method. Intraspecific hybridization was investigated using two stocks of S. broughtonii; the China population (C) and the Korea population (K). The shell length and wet weight were compared between two reciprocal hybrid crosses (C♀ × K♂, CK and K♀ × C♂, KC) and two parental groups (C♀ × C♂, CC and K♀ × K♂, KK) during the adult culture stage. The results showed that the shell length and wet weight of the KC group were significantly higher than those of the other three groups. The heterosis rates of the CK group for shell length was between –2.03% and 5.44%, and the KC group was between 4.40% and 14.74%. The heterosis rates of the CK group for wet weight was between 1.41% and 7.71%, and the KC group was between 5.32% and 23.71%. The heterosis rates of the KC group for shell length and wet weight was higher than those of CK group. The wet weight of the CK group was significantly higher than that of the CC and KK groups at 14 and 15 months, respectively. Additionally, the heterosis rate of the two reciprocal hybrid crosses for wet weight was positive and high. In summary, the heterosis rates of the KC group for shell length and wet weight were markedly higher than those of other three groups, thus, the KC hybrid group could be selected as an ideal breeding material.

Abstract:HSP70s are heat shock proteins that exist widely in prokaryotes and eukaryotes, and play important roles in biological resistance. The full cDNA sequence of a hsp70 gene in Saccharina japonica (Sjhsp70) of 3778 bp was obtained by RACE technology. Sjhsp70 consisted of an open reading frame (ORF) of 2892 bp, a 5ʹ untranslated region (UTR) of 101 bp, and a 3ʹ UTR of 785 bp. Analysis of amino acid content and physicochemical properties showed that SjHSP70 is a stable protein. Comparison between the amino acid sequences of SjHSP70 and the well-characterized orthologous HSP70 proteins from various species indicated that SjHSP70 shares the highest similarity with the orthologous protein from Ectocarpus siliculosus. The phylogenetic tree of the examined proteins indicated that SjHSP70 formed a group with a HSP70 from E. siliculosus. Secondary structure analysis of SjHSP70 showed that it is composed of 35 α-helices and 25 β-strands. Three-dimensional homology structure modeling of SjHSP70 was predicted by the I-TASSER server according to the 3C7N template. Overall, the folding and secondary structures of SjHSP70 were highly similar to those of 3C7N. To study the correlation between SjHSP70 and the resistance to high-temperature stress in S. japonica, real-time variation in the expression of Sjhsp70 under different temperatures and different stress times was determined. The results showed that high temperature had a significant effect on the expression of Sjhsp70. The expression of Sjhsp70 continued to increase with increase in temperature, with the maximum Sjhsp70 expression at 25℃ after 24 h of high-temperature stress. This result indicates that Sjhsp70 is probably related to high-temperature resistance in S. japonica. This study provides a foundation for the characterization of SjHSP70 and for the study of SjHSP70 function in the high-temperature stress response in S. japonica.

Abstract:To investigate the effect of nitrogen sources (NO3-N, NH4+-N, and Urea) on the physiological characteristics of Sargassum hemiphyllum seedlings, the seedlings were cultured for 24 days in different concentrations of each nitrogen (N) source, and the growth rate and biochemical composition were determined. The results showed that the N source and N concentration had significant effects on the growth rate and biochemical composition of the seedlings. With increasing N concentrations, the relative growth rates and tissue N contents of the seedlings increased, and when tissue N contents approached or reached N requirement (0.032%/d) for the maximum growth rate in 25~150 μmol/L, the relative growth rates reached the maximum at 50 μmol/L. The maximum relative growth rate under Urea enrichment was significantly lower than that under NO3-N and NH4+-N enrichments. The relative growth rate of the seedlings in 10 and 25 μmol/L NH4+-N was significantly higher than that in the same concentration of NO3-N, but the relative growth rate in 50~150 μmol/L showed an opposite change. Except for the highest N concentration (150 μmol/L), the photosynthetic pigments, soluble protein, and tissue nitrogen contents gradually increased, while the soluble sugar content gradually reduced as N concentration increased. At the same N concentration, the contents of soluble sugars, chlorophyll a, and chlorophyll c of seedlings were the highest under NO3-N enrichment, the contents of the soluble protein content and tissue N content were the highest under NH4+-N, and the fucoxanthin content was the highest in Urea-enriched culture medium. When the NH4+-N concentration reached 150 μmol/L, a significant decrease in growth and soluble protein content was observed, indicating the presence of a stressful condition. In conclusion, culture medium enriched in NO3-N (50~150 μmol/L) or NH4+-N (25~100 μmol/L) promoted the growth, photosynthesis, and dry-matter accumulation of S. hemiphyllum seedlings, suggesting that the seedlings may be successfully cultured indoors during summer.

Abstract:Fishmeal is an important animal protein feed that is rich in protein and vitamins and is a major raw material of animal feed. Arsenic is an essential element for animal growth but is also a cumulative toxic element. Arsenic poses a great threat to animals when its concentration exceeds a certain limit. Serious environmental pollution has caused an increase in the level of arsenic in the environment, and thus, the content of arsenic has increased in fish. This can directly affect the quality of fishmeal, with unfavorable effects on food safety. Therefore, it is crucial to accurately determine the content of total arsenic in feed stuffs. The accurate determination of total arsenic in samples depends on pre-treatment methods. At present, the major pre-treatment methods for arsenic include dry-ashing, wet digestion, and microwave digestion, but none of these methods can completely convert the speciation of arsenic to arsenic ion. Dry-ashing leads to the loss of arsenic due to high temperature and spatter. Wet digestion and microwave digestion do not convert complex forms of arsenic to arsenic ion because of temperature, acid type, and dosage. A novel method was established for the quantitative determination of total arsenic in fishmeal by atomic fluorescence spectrophotometry after microwave dry-ashing mixed digestion. Fishmeal was digested in a microwave, ashed, and the arsenic content was detected by atomic fluorescence spectrophotometry. Recovery was in the range 96.8%~103.6%, the relative standard deviation was 2.4%, and the detection limit was 0.1 μg/L. Results of China National Accreditation Service for Conformity Assessment proficiency testing were in accordance with the median. The results indicated that microwave dry-ashing mixed digestion can reduce the loss associated with the high-temperature of ashing and incomplete microwave digestion, and convert different forms arsenic into arsenic ion. This effective, sensitive, and reproducible method can be used to determine total arsenic levels in fishmeal and other complex samples containing arsenic forms.

Abstract:Kisspeptin (Kiss or Kp), a novel physiologically active peptide encoded by the KISS1/Kiss1 gene, activates its cognate receptor KissR (also known as GPR54) in various target tissues to exert disparate functions, including inhibition of tumor metastasis and control of reproductive function. The KISS1 gene was originally isolated from human melanoma and breast cancer cells, and kisspeptin was initially called metastin in consideration of its suppressive effects on tumor growth and metastasis. With the exception of the platypus, a mammalian monotreme, which has two forms of kisspeptin genes (Kiss1 and Kiss2), there is only one ligand, Kiss1, and its receptor, Kiss1R in placental mammals. However, this situation is different and even complex in non-mammalian species. Three kiss/kissr genes were described in amphibians, while searches in the chicken genome databases failed to identify these paralogous genes. To date, multiple forms of kiss/kissr genes have been identified in many teleosts, including Cypriniformes, Perciforms, Pleuronectiforms, Tetraodontiforms, Beloniforms, Scorpaeniformes, Salmoniformes and Gadiformes. A dual kisspeptin system, kiss1/kiss1r and kiss2/kiss2r, have been detected in medaka, zebrafish, goldfish, chub mackerel, striped bass, and European sea bass, while only kiss2/kiss2r was identified in orange-spotted grouper, grass puffer, Nile tilapia, Atlantic halibut, Senegalese sole, and half-smooth tongue sole. In addition, the physiological relevance and functions of the Kiss/KissR system for the neuroendocrine regulation of reproduction remains to be established in fish. It should be noted that the mechanisms underlying the actions of Kiss on the hypothalamo-pituitary-gonadal (HPG) axis are still far from being fully understood. Given the multiple forms of kiss and kissr genes obtained in teleosts, the regulation of fish reproduction by the Kiss system is even complex. This review briefly summarized the progress of research on Kiss and its receptors, with special emphasis on the physiological functions of Kiss in fish, the signaling transduction pathways as well as the regulation of kiss/kissr gene expression. We hope that this review will contribute to future studies.