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:Studies of the food web are an important inlet to carry out integrative research on sustainable marine ecosystems. These investigations further highlight the main food chain in the study of end-to-end food webs, and the research is conducted at each trophic level (TL). Under multiple pressures of human activities and climatic change, coastal sea ecosystems have undergone significant changes. Therefore, it is very important to clarify the food contact and its present variation in the Bohai Sea ecosystem. In this study, we analyzed 27 different fish species with 10156 stomachs, collected from bottom trawl surveys during 2010~2011 in the Bohai Sea. The percentage frequencies of occurrence, number, and weight of different prey categories were calculated to describe the diet composition. The Shannon–Wiener diversity index was used to evaluate the dietary breadth of each fish. The TL of each fish was calculated, and then the fish were classified as low, medium, and high TL (≤3.8, 3.9~4.4, and ≥4.5, respectively). The results showed that the 27 fish species, including 12 low TL species (Clupanodon punctatus, Sardinella zunasi, Enedrias fangi, Thrissa kammalensis, Callionymus beniteguri, Cynoglossus joyneri, Cynoglossus lighti, Setipinna taty, Enchelyopus elongates, Chaeturichthys stigmatias, Trichiurus muticus, and Chaeturichthys hexanema), 12 medium TL species (Scomberomorus niphonius, Pseudosciaena polyactis, Hexagrammos otakii, Johnius belangerii, Sebastiscus marmoratus, Sebastodes fuscescens, Liparis tanakae, Platycephalus indicus, Hemitripterus villosus, Argyrosomus argentatus, Chelidonichthys kumu, and Lateolabrax japonicus) and three high TL species (Saurida elongate, Lophius litulon, and Sphyraena pinguis), were classified into omnivores, zooplanktivores, benthivores, mixed animal predators, and piscivores. Compared to that in the 1990s, the TL of each fish has not changed much. Three kinds of high TL fish belonged to the piscivores fish category, and their dietary breadth was very low, and so they were stenophagous fish. Finespot goby and small yellow croaker had the highest dietary breadth and were the most important prey species in the food web of the Bohai Sea ecosystem; therefore, their euryphage was beneficial to the material and energy flow of each trophic hierarchy in the food web. Presently, the food chain “zooplankton–anchovy–large piscivores fish” was basically destroyed, and so, the main food chains changed to “plants, organic detritus–Alpheidae– fishes” and “benthic animals–goby fish, small yellow croaker–large commercial fishes” in the food web of the Bohai Sea. The weakening of the pelagic food chain is not conducive to the carbon sink of the marine ecosystem and the material and energy flow of the food web. Therefore, studies of the end-to-end food web and the main food chain of the Bohai Sea should change accordingly.

Abstract:An accurate grasp of species distribution is a requirement for biodiversity studies, and understanding species’ biomass is crucial to assessing ecosystem productivity and material cycles. Traditional bioassays and biomass assessments are based on data from trawl surveys. However, trawl surveys often do not provide a true reflection of species distribution and resource status because of differences in the living habits and resource status of different species. To accurately grasp species distribution and resource status, it is imperative to identify cost-effective aquatic organism survey methods to provide technical support for the research and protection of aquatic ecosystem biological diversity. In recent years, the rapid development of molecular biology research has transformed species identification methods from traditional morphological classifications to molecular biology-based techniques, especially the development and application of environmental DNA technology. Environmental DNA is a ubiquitous free radical released from the skin, mucus, saliva, sperm, secretions, eggs, feces, urine, blood, roots, leaves, fruits, pollen, and decaying bodies of DNA molecules. Environmental DNA technology refers to the method of qualitatively or quantitatively analyzing DNA fragments directly from environmental samples (such as soils, sediments, and water bodies) using sequencing techniques. Environmental DNA technology has become a novel method of aquatic organism survey, which is mainly used in processes such as the prevention and control of biological invasion, protection of endangered species, and evaluation of biodiversity and biomass. Here, we review the development of environmental DNA technology, the operational flow, and its application in aquatic ecosystems, as well as the advantages and existing problems. In addition, we project the prospect of environmental DNA in the field of ecology. We hope that this review will provide novel ideas and methods for the research and protection of the biological diversity of aquatic ecosystems to ensure that aquatic resources can be fully utilized.

Abstract:The stable nitrogen isotope ratio (δ15N) has been widely used in the analysis of marine ecosystem food webs and nutrient dynamics. It provides a powerful tool for estimating the trophic positions of consumers in food webs. Stable isotope analysis of fishes is often performed using muscle samples to study trophic position relationships as well as food webs in marine ecosystems; however, it requires the sacrifice of fishes. While few studies have reported the composition of stable isotopes in the different tissues and organs, fewer domestic studies have reported the δ15N value in the viscera, otolith, gill, fin, and scale to evaluate diet information, trophic position relationships, and food web analyses of ecosystems, and notably, the composition and measurement of the stable isotopic. The δ15N values of the muscle, viscera, otolith, gill, fin, and scale were determined by isotope ratio mass spectrometry, and statistical analysis was performed. The δ15N distribution characteristics in different tissues of small yellow croaker (Larimichthys polyactis) were investigated. The results showed that the δ15N values between the viscera, gill, fin, scale, and muscle were not significantly different (P>0.05). The highest δ15N levels was observed in the muscle followed by the scale, fin, viscera, and gill; and the average value ranged from 10.29‰ to 10.72‰. However, the δ15N value in the otolith was much lower than that in other tissues and was significantly different among other tissues and organ samples (P<0.01), and the average was 7.08‰. These results indicated that the otolith was different from the other tissues. Pearson correlation analysis showed that the correlation of δ15N between different tissues and the muscle was strong, and the correlation coefficient was more than 0.72. It is feasible to use tissues other than the muscle to study marine ecosystem dynamics. Our findings advocate the analysis of δ15N in tissues and organ to establish trophic level, food web structure, and mean trophic level in aquatic ecosystems.

Abstract:Edwardsiella tarda is one of the pathogenic bacteria of Paralichthys olivaceus that seriously affects the sustainable development of flounder aquaculture. In the present study, we used the family of high-quality flounder developed in 2012, 2013, and 2014 as the parents, and developed 28 flounder families in 2016, including 4 F3 generation families, 23 F4 generation families, and control family. After 60 days of growth trait assay and artificial bacterial infection experiments, seven families with strong resistance to disease (survival rate > 66%) were screened. F1639 was screened as the F4 generation family with enhanced disease resistance and fast growth rate. Furthermore, its survival rate was 77.23%, which was 32.75% higher than that of the average of 28 families. The daily growth rate was 0.174 cm/d, which was 0.023 cm/d higher than that of the other 28 families. F1639 was derived from the 2007 (F0750, resistant to Vibrio anguillarum family), 2009 (F0927, resistant to V. anguillarum family), and 2014 (F1421, resistant to E. tarda family) families. The results showed that the disease resistance of the breeding families improved. Furthermore, the breeding ability of the offspring can be improved by the breeding of the family (the selection of compatriots). The present study has laid a foundation for breeding new variety of Japanese flounder with higher disease resistance and growth rate. Furthermore, it has important potential application in the genetic breeding for disease-resistant and fast growth stock in other fish species.

Abstract:Arginase Ⅱ(Arg-Ⅱ) is a type of arginase. It mainly participates in the urea cycle, and has been found to play an important role in the pathological process. However, its role in fish is not well reported. In this milieu, we obtained the full cDNA of Arg-Ⅱ of Japanese flounder (Paralichthys olivaceus) and detected its space-time expression profile in the immune tissue and bacterial infected tissue of Japanese flounder. The results showed that the Arg-Ⅱ gene consists of 1882 nucleotides, including an open reading frame (1150 bp) that encodes a protein of 349 amino acids (with deduced molecular mass of 38.02 kDa and theoretical isoelectric point of 6.42). The amino acid of Arg-Ⅱ protein in Japanese flounder displayed the typical structure of arginase, therefore we assumed that it might have functions similar to that in mammals. Phylogenetic tree analysis also confirmed that fish Arg-Ⅱ formed a cluster, and Japanese flounder Arg-Ⅱ was most closely related to that of Lates calcarifer (93%). Quantitative real-time PCR (q-RT PCR) analysis showed that the mRNA of Arg-Ⅱ is mainly expressed in immune tissues, such as the liver, spleen, gills, and kidney. Intensive study showed that the mRNA expression of gene Arg-Ⅱ was significantly upregulated after 6~12 h of Edwardsiella tarda challenge, and then returned to normal condition in the immune tissues of Japanese flounder. The expression of Arg-Ⅱ was also significantly upregulated in the in vitro cultured E. tarda challenged Japanese flounder phagocyte. The present study demonstrates that the gene Arg-Ⅱ might participate in the immune response of Japanese flounder to E. tarda infection, thus indicating that the gene Arg-Ⅱ might play an important role in the antibacterial immunity of Japanese flounder.

Abstract:The innate immune system acts as the first defense against invading pathogens through three classes of pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs), including the Toll-like receptors (TLRs), RIG-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). The nucleotide-binding oligomerization domain-like receptors are a family of innate immune receptors that link cytosolic sensing of microbes and danger stimuli to activate immune responses. As a member of the NOD-like receptor family, NOD2 can recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). In the present study, the Japanese flounder (Paralichthys olivaceus) NOD2 (PoNOD2) was identified by searching the transcriptome sequence of Japanese flounder based on the available NOD2 cDNA sequences of other fishes. The complete open reading frame (ORF) of PoNOD2 is 2964 bp and encodes a polypeptide of 988 amino acids. The gene PoNOD2 is composed of three main domains: an N-terminal domain containing two adjacent CARDs, a central NACHT domain, and a multiple C-terminal LRRs. The phylogenetic tree analysis suggests that the gene PoNOD2 is closely related to that of the Fugu rubripes (Takifugu rubripes). Tissue expression analysis by qRT-PCR showed that the gene PoNOD2 is constitutively expressed in all the examined tissues, predominantly in the spleen and liver. Real-time PCR showed that the infection of Edwardsiella tarda can significantly upregulate the expression of PoNOD2 in the tissue of Japanese flounder. In vitro immune stimulation experiments showed that PoNOD2 expression could be upregulated by peptidoglycan, polyinosinic: polycytidylic acid, and E. tarda infection. Subcellular localization of the PoNOD2 protein is in the cytoplasm of the gill cells of flounder. When the flounder gill (FG) cells were infected by E. tarda, the overexpression of PoNOD2 can inhibit the bacterial replication and upregulate the transcription of pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8. These results suggest that PoNOD2 plays a key role in the resistance of P. olivaceus to E. tarda infection and in the modulation of immune responses.

Abstract:The aim of this study was to study the effect of dietary β-glucan on certain growth and blood physiological indices of the rainbow trout (Oncorhynchus mykiss). The experimental animals were robust, with similar weight of (450±50) g. The control group was fed basal feed, and the experimental groups were fed basal feed supplemented with β-glucan at three concentrations: 0.05%, 0.1%, and 0.2%, three replicate tanks for each group. After 30 days of feeding, the weight gain rate (WGR) in the 0.2% β-glucan group was the highest, significantly higher than that of the other groups (P<0.05). Meanwhile, the WGR of the 0.05% β-glucan group was the lowest. The specific growth rate (SGR) of the 0.2% β-glucan group was significantly higher than that of the other groups (P<0.05). No significant differences in SGR were found among the 0.05% and 0.1% β-glucan groups and the control group (P>0.05). The hepatosomatic index (HSI) of the 0.2% β-glucan group was the highest, significantly higher than that of the other groups (P<0.05). The condition factor (CF) of the 0.2% β-glucan group was significantly lower than that of the other groups (P<0.05). After 30 days of feeding, the number of leukocytes and erythrocytes and the plasma hemoglobin concentration were significantly higher in the experimental groups than in the control group (P<0.05). The number of leukocytes in the 0.05% β-glucan group, which was not significantly different from that of the 0.1% β-glucan group (P>0.05), was significantly higher than that of the 0.2% β-glucan group and control group (P<0.05). The number of erythrocytes in the 0.1% β-glucan group was the highest, significantly higher than that in the other groups (P < 0.05). Furthermore, the number of erythrocytes in the β-glucan groups was significantly higher than that of the control group (P<0.05). The changes in hemoglobin concentrations were consistent with changes in erythrocyte count. In conclusion, dietary β-glucan can improve the growth performance and favorably change the blood physiology indicators of rainbow trout, with the appropriate concentration of β-glucan being 0.2%.

Abstract:The present study aimed to investigate the effects of dietary protein level on the growth performance, feed efficiency, and immuno-enzymatic activity in Paramisgumus dabryanus ssp. Four diets were formulated with 25%, 30%, 35%, and 40% protein, respectively. In total, 720 P. dabryanus ssp with an initial body weight of (8.57±0.35) g were randomly divided into 4 groups with 3 replicates per group and 60 fish per replicate. The cultivation of P. dabryanus ssp lasted for 60 d feeding with the four diets. The results showed that the final weight (FW), specific growth rate (SGR), and feed efficiency rate (FER) of P. dabryanus ssp increased with increase in dietary protein level, and plateaued at the dietary protein level of 35%. Initially, the protein efficiency ratio (PER), protein retention (PRE), and survival rate (SR) increased, and then started to decrease. However, the feeding rate (FR) showed a gradual decrease with increasing dietary protein level. The results of broken line simulation of FW, SGR, and FER showed that the P. dabryanus ssp had the optimal growth and feed efficiency rates at a dietary protein levels between 34.57% and 35.37%. Furthermore, the quadratic polynomial regression analysis revealed that the utilization of protein by the P. dabryanus ssp reached the highest level at the dietary protein levels between 33.61% and 34.68%. The activity of superoxide dismutase (SOD) in P. dabryanus ssp increased at first and then plateaued with increasing dietary protein level. The activity of catalase (CAT) in the P. dabryanus ssp increased at first and then decreased. The activity of glutamic oxaloacetic transaminase (GOT) and glutamate pyruvic transaminase (GPT) in the P. dabryanus ssp increased at first and then decreased with increase in dietary protein levels. These results show that a suitable dietary protein level can improve the growth rate, feed efficiency, and immuno-enzymatic activity of P. dabryanus ssp. Furthermore, a comprehensive analysis of growth performance, feed efficiency, and immunity-related enzyme activity revealed that the optimal dietary lipid level for P. dabryanus ssp is 34.68%~35.37%.

Abstract:As an eco-friendly wastewater treatment method, constructed wetlands have been widely used in purifying aquaculture wastewater. However, due to the effects of salt stress of seawater, the feasibility of treating mariculture wastewater in constructed wetland with different plants needs further study. To investigate the water contaminant removal efficiency, two integrated vertical flow constructed wetlands (IVCW) were constructed. Phragmites australis and Spartina alterniflora were selected as the constructed wetland plants. The salinity tolerance of P. australis was significantly different from that of S. alterniflora. The content of ammonia nitrogen (NH4-N), nitrite nitrogen (NO2-N), nitrate nitrogen (NO3-N), phosphorus, total phosphorus (TP), and potassium permanganate index (CODMn) were determined for 14 d. The two constructed wetland were filled with fine sand, vermiculite, and corallite. The results showed that the optimum salinity of P. australis was 20 and that of S. alterniflora was less than 35. The physical and chemical properties of the purified water were: temperature (28.60±0.71)℃, pH 7.60±0.04, and dissolved oxygen (DO) (5.32±0.45) mg/L. This demonstrated that the efficiency of P. australis wetland and S. alterniflora wetland in the removal of NH4-N and CODMn was (85.48±0.50)% and (57.74±4.40)%; and (95.01±1.18)% and (72.84±2.64)%, respectively. The efficiency in the removal of phosphate and TP were 40.00%~50.00% by both the constructed wetlands. These results suggested that the salt tolerance of S. alterniflora was better than P. australis. There were significant differences in NH4-N and CODMn between P. australis wetland and S. alterniflora wetland (P<0.05). However, the difference between the wetlands was not significant for phosphate and TP (P>0.05). In the short term, the efficiency of the removal of NH4-N and CODMn from mariculture effluents by S. alterniflora wetland was better than by the P. australis wetland. Under high-salinity stress, S. alterniflora had strong salt tolerance and grew better in seawater, and had better purification efficiency than P. australis. However, the efficiency in the removal of phosphate and TP exhibited no significant difference between the two wetlands.

Abstract:To optimize recirculating aquaculture systems (RAS), we performed a water-quality detection experiment to study the effect of clearance rates of ammonia nitrogen, chemical oxygen demand (COD) and suspended solids (SS) by a biological filter in RAS under conditions of ammonia nitrogen and dissolved oxygen load. The experimental results for ammonia nitrogen load showed that the ammonia nitrogen concentration of the inlet water had a significant effect on the ammonia nitrogen (positive correlation) and COD (positive correlation) of the outlet water (P<0.01), and did not have a significant effect on SS. The optimal treatment effect of the water was observed when the ammonia nitrogen concentration of the inlet water was 0.45~0.65 mg/L (average clearance rates of ammonia nitrogen, COD, and SS were 82.1%±3.3%, 7.1%±1.5%, and 5.8%±1.6%). Experimental data for the dissolved oxygen load showed that dissolved oxygen concentration had a significant effect on ammonia nitrogen (negative correlation) and COD (negative correlation) treatment (P<0.05), and did not have a significant effect on SS. The optimal treatment effect of the water was observed when the dissolved oxygen concentration was 5.0~7.0 mg/L, (average clearance rate of ammonia nitrogen, COD, and SS were 78.7%±3.5%, 23.0%± 5.3%, and 7.1%±2.0%). Therefore, when the concentration of ammonia nitrogen is 0.45~0.65 mg/L, the concentration of dissolved oxygen is 5.0~7.0 mg/L. The treatment effect of ammonia nitrogen, COD, and SS is optimized by the coral biofilter of RAS in this experimental environment.

Abstract:In the present study, a preliminary evaluation was conducted on the cold tolerance and growth traits of 69 Fenneropenaeus chinensis by indoor artificial cooling. A linear mixed model with average information restricted maximum likelihood was used to evaluate the genetic parameters. Two animal models were used to evaluate the heritability of body weight (BW) and body length (BL) under low-temperature stress in F. chinensis. The heritability of BW and BL observed in model 1 were (0.206±0.177) and (0.187±0.179), respectively, and for model 2, these were (0.317±0.065) and (0.298±0.063), respectively, which ranged between medium to low. The differences between the two models were tested by likelihood ratio test, and the likelihood ratio values for Model 1 and Model 2 were 1.640 and 1.764, respectively, which were not significantly different (P>0.05). The results showed that the two models were not significantly different, and that model 2 was the optimal model. The heritability of the survival status at half lethal time (SS50) among cold tolerant traits was (0.169±0.078). The phenotypic and genetic correlation coefficients between BW and BL under low-temperature stress were (0.823±0.010) and (0.969±0.018), respectively, which were relatively high. The phenotypic correlation coefficients between BW and SS50; and BL and SS50 were comparatively low with values (0.187±0.030) and (0.218±0.030), respectively. The genetic correlation coefficient between BW and SS50; and BL and SS50 were comparatively high with values (0.517±0.205) and (0.538±0.203), respectively. The results showed that during breeding of F. chinensis, the cold tolerant variety can be selected together with growth traits.

Abstract:Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome (EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16S rRNA and groEL gene sequence. The serotype of the bacterium is O1:KUT (K untypeable). It revealed positive amplification of the genes pirAVP and pirBVP which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose (LD50) value of 7.96×103 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100% died at 24 h. Subsequent histological analyses showed that the hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells, which was the typical pathological characteristics of AHPND. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.

Abstract:Acute hepatopancreatic necrosis disease (AHPND) has pathogenic diversity causing high mortality and economic losses in shrimp culture. We isolated PV130903A (Vibrio parahaemolyticus), PV140731A (V. harveyi), PV150526A (V. alginolyticus), and PV140821A (V. sp. Ex25) from the infected Litopenaeus vannamei, and designed a germicidal experiment on the isolated pathogens. Polyhexamethylene guanidine (PHMG), hydrogen peroxide (H2O2), povidone-iodine (PVPI), and chlorine dioxide (ClO2) were chosen to determine their concentration, killing rate, and killing time on the four pathogenic bacteria. Furthermore, an experiment was also conducted to determine the killing efficiency of PHMG on the Vibrios in the pond water. The results showed that 2 μl/L ClO2 killed all the bacteria in 1 h; 1 μl/L PHMG and H2O2 killed all the bacteria in 2 h; and 6 μl/L PVPI killed all the four bacteria in 6 h. It is noteworthy that the killing rate of PHMG and PVPI gradually increased with time. In contrast, the killing rate of H2O2 and ClO2 decreased after attaining the peak value. In the experiment carried out on the Vibrios in the pond water, 0.5 μL/L, 1 μL/L, and 2 μL/L PHMG decreased the total amount of Vibrios in the water to the minimal value of 79.14% on day 4, 82.48% on day 3, and 87.30% on day 3, respectively. Furthermore, the total amount of Vibrios in all the three concentration groups recovered slowly after reaching the minimal value. On day 11, the total amount of Vibrios in the three groups was still lower than the initial value of 3.58%, 5.53%, and 6.10%, respectively. The bactericidal ability of these four disinfectants was in the order: PHMG > H2O2 > ClO2 > PVPI. Based on the bactericide concentration, germicidal efficacy, duration, and cost, it was proved that PHMG has be efficiently used in aquaculture and it has a good application prospect in the future.

Abstract:The present study was conducted to evaluate the differences between the growth and reproductive molting of Exopalaemon carinicauda. In this milieu, we analyzed the differences in the representative stages (intermoult C, premoult D, and postmolt AB) of the two types of molting in relation to three parameters: non-specific immune indexes including phenoloxidase (PO), superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), molting-related enzyme (Chitinase), and ecdysone concentration (MH). The activity of PO during different stages of growth molting exhibited no significant difference. While the activity of PO during reproductive molting decreased at first and then increased, and showed significant differences among different stages. At the intermoult stage, the activity of PO of growth and reproductive molting was not significantly different. At the premoult stage, the activity of PO of reproductive molting was significantly lower than that of growth molting. At the postmoult stage, the activity of PO of reproductive molting was significantly higher than that of growth molting. During growth molting, the activity of SOD at all the three stages gradually increased, and demonstrated significant differences. During reproductive molting, the activity of SOD at the postmoult stage was significantly lower than that at the other two stages, and the difference between the intermoult and premoult stages was not significant. At the intermoult stage, the activity of SOD of reproductive molting was significantly higher than that of growth molting. At the premoult stage, there was no significant difference between them, while at the postmoult stage, the SOD activity of reproductive molting was significantly lower than that of growth molting. During growth molting, the activity of AKP and ACP at all the three stages had the same trend, that is they increased at first and then decreased, and the difference was significant. The activity of enzymes during reproductive molting had the same trend as those during growth molting. However, the activity of enzymes at all the three stages during growth molting was significantly higher than that during reproductive molting. At different stages of growth molting, chitinase activity and MH hormone concentration had the same trend, that is they gradually increased, and the difference of the stages was significant. Furthermore, during reproductive molting, they decreased at first and then increased, and the differences between the stages were significant. During the intermoult and premoult stages, chitinase activity and MH hormone concentration of growth molting was significantly lower than those of reproductive molting. During the postmoult stage, chitinase activity and MH hormone concentration of growth molting were significantly lower than those of reproductive molting. The present study conducted is a preliminary study on growth and reproductive molting. The results showed that there was a significant difference between the growth and reproductive molting because of the ovary development. The results provide a theoretical base and a scientific ground for further research on the mechanism of molting and artificial breeding.

Abstract:In the present study, full-length complementary DNA (cDNA) of Drosha and Exportin 5 involved in the pathway of microRNA (miRNA) biogenesis of the swimming crab Portunus trituberculatus were cloned by rapid amplification of cDNA ends (RACEs). The length of the Drosha gene is 3443 bp, which is predicted to encode a polypeptide of 1038 amino acids, containing two ribonuclease (RNase) Ⅲ domains (RⅢDa and RⅢDb) and a double-stranded RNA binding domain (dsRBD). The full-length of Exportin 5 is 5000 bp, which is predicted to encode a polypeptide of 1208 amino acids, containing an importin-β N-terminal domain (IBN-N) and an exportin 1-like domain (XPO-1). Homology analysis revealed that Drosha in P. trituberculatus is highly similar to those in some species and shares the highest similarity with that in Marsupenaeus japonicus (94%). Quantitative real-time PCR (qRT-PCR) analysis showed that Drosha and Exportin 5 are expressed in all the tested tissues, and were highly expressed in the ovary and hepatopancreas tissues (P<0.05). At different stages of testis development, the expression of the genes Drosha and Exportin 5 shared the same trend, and both showed the highest expression at stage Ⅱ. During ovarian development, the expression level of the genes Drosha and Exportin 5 increased gradually from phase Ⅱ to phase Ⅴ. The results suggest that the genes Drosha and Exportin 5 cooperatively regulate the gonadal development of P. trituberculatus in an miRNA-dependent manner. Furthermore, the results provide useful information for studies on the regulation of gonadal development in P. trituberculatus.

Abstract:The accessory nidamental gland (ANG) is a type of accessory gland found in Sepioidea species. It is an important part of the female reproductive system and plays an important role in the formation of the egg membrane, disease control, and buoyancy adjustment. Here, the ultrastructure of the ANG from adult Sepia esculenta was studied using light and electron microscopy. The results showed that the ANG forms an ear-like shape; its bottom forms an ‘M’ shape, which is closely attached to the nidamental gland; and the color is white and orange. ANG is composed of a gland wall, tubules, and connective tissue. The inside layer is muscular, whereas the outer layer is composed of simple columnar epithelium cells. A number of tubules were observed with an average diameter of 70 μm; the lumen surface has a microvilli layer, and a large number of substances are secreted inside the lumen. The connective tissue contains concentrated distributions of blood vessels and muscle fibers. The cytoplasm of the epithelial cells contains many concentrated distributed vesicles, and some contain secretion globules. The microvilli are widely distributed; while the cilia are discontinuous, with some concentrated and others arranged in a single line. The tubule lumens contained a large number of symbiotic bacteria, most of which were coccoid-shaped bacteria, containing many secretion globules. Consequently, the ANG of S. esculenta possesses secretory function; the existence of symbiotic bacteria in the ANG might play an important role in disease resistance during embryonic development. We show the ultrastructure of the ANG in adult S. esculenta and discuss its function and role in reproduction. The aim is to reveal the breeding strategies of S. esculenta and to enrich knowledge of the reproductive biology of cephalopods, thus, providing a reference for optimization of S. esculenta breeding technology.

Abstract:According to the transcriptome database of Sepiella japonica, the microsatellites have been explored at a high-throughput scale using a MIcroSAtellite identification tool. In total, 127575 unigenes were obtained after assembly and removal of redundant sequences. The total length of unigenes after sequence splicing was 103104058 bp. The microsatellites in the unigenes were analyzed, and a total of 62124 simple sequence repeats (SSR) (complete SSR, 36813) was identified in 50626 unigenes, accounting for 48.70% of the total unigenes, with one SSR per 16.6 kb. In addition, the microsatellite sequences mainly composed of dinucleotide repeats (31227, 50.27%), followed by trinucleotide repeats (16230, 26.13%). Furthermore, 122 types of repeat motifs were classified in all SSR motifs, (AT/AT)n was the most frequent repeat motif (23.33%), followed by (AAAG/CTTT)n (17.42%). The average length of microsatellites (complete SSR) was 25.87 bp, while that of the microsatellites over 20 bp was 51%. There was a significant negative correlation (P<0.01) between the frequency of microsatellites and the length, the correlation coefficient was ‒0.594. The results of the present study lay a foundation for the development of highly polymorphic microsatellite primers to identify parentage relationship and population genetic diversity, and to evaluate the effect of enhancement and release of S. japonica.

Abstract:Amphioctopus fangsiao, a synonym of Octopus ocellatus in China, is one of the most important economic species in the northern coast of China. Owing to overfishing and ever-increasing market demand, the wild resources of A. fangsiao are decreasing significantly, and therefore artificial breeding is imperative. The present study on the reproduction model of A. fangsiao would provide basic data for artificial breeding and germplasm conservation of the species. In the present study, 4 groups of triplex PCR sets were established using 12 high polymorphic microsatellite markers developed in our laboratory. Through the identification of paternity of 4 female parents (♀1, ♀2, ♀3, and ♀4), 104 offspring and 19 candidate male parents of the reproduction model A. fangsiao were analyzed. The results showed that the four groups of microsatellite multiplex PCR sets worked effectively in the paternity relationship analysis, and the identification rate of 104 offspring was 94.23%. Twelve offspring and 3 males were identified in ♀1 group, and the number of offspring and males identified in the groups ♀2, ♀3, and ♀4 were 40 and 7; 19 and 2; and 27 and 4, respectively, and ♂2, ♂3, ♂7, and ♂10 mate with two or more females, which indicates that A. fangsiao is polygynandry. Polygynandry is an adaptive mechanism by aquatic animals to environmental changes and human fishing pressure. It can effectively improve the genetic diversity of alleles and bring about genetic variation among offspring. In addition, polyandry plays a positive role in maintaining the stability of the population structure of A. fangsiao, and in improving the reproductive efficiency and offspring quality. The offspring from four groups assigned to ♂2 were as high as 71.43% (10/14), 60.98% (25/41), 85.71% (18/21), and 75% (21/28), respectively, which was higher than that assigned to the other male parents. This indicates that most of the sperm involved in fertilization were from ♂2; thus, ♂2 takes advantage of sperm competition and cryptic female choice. The present study provides important data for artificial breeding and proliferation, and also for the release of A. fangsiao.

Abstract:In the past thirty years, global aquaculture production continues to grow at an annual rate of nearly 9%. The rapid development of global aquaculture not only helps maintaining world food supply, but also brings environmental pollution, biodiversity decline and a series of ecological problems, causing widespread concern in the international community. The issue of aquaculture carrying capacity, that is, the carrying capacity of resource and environment for aquaculture, has become a worldwide concern and a key scientific question for sustainable aquaculture development. In this paper, evolvement of the concept of aquaculture carrying capacity and environmental carrying capacity are elucidated briefly, exemplary work on aquaculture carrying capacity are introduced, the development of assessment methods and modeling of aquaculture carrying capacity are analyzed, and its related issues and prospective application in aquaculture governance are discussed in order to promote carrying capacity-based aquaculture spatial planning. Scientific evaluation and comprehensive application of aquaculture carrying capacity may solve the environmental problems constraining the sustainable development of mariculture to a certain degree, and may provide scientific basis for ecosystem-based aquaculture spatial planning and governance.

Abstract:Globally, infectious hypodermal and haematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimp, causing huge economic loss to the shrimp farming industry. Further, IHHNV has been listed as a reportable crustacean disease pathogen by the World Organization for Animal Health (OIE). It is widely distributed in China and has become an important virus affecting shrimp culture. In the present paper, IHHNV pathogenicity-related progress geographically, host range, pathogenicity type, pathogenicity in host at different growth stages, interference with white spot syndrome virus (WSSV), and pathogensis mechanism of IHHNV were reviewed. The paper aimed to provide systematic references for the studies on control of IHHNV.