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Despite the prevalence of research on single-subject cerebral morphological networks in recent years, whether they can offer a reliable way for multicentric studies remains largely unknown. Using two multicentric datasets of traveling subjects, this work systematically examined the inter-site test-retest (TRT) reliabilities of single-subject cerebral morphological networks, and further evaluated the effects of several key factors. We found that most graph-based network measures exhibited fair to excellent reliabilities regardless of different analytical pipelines. Nevertheless, the reliabilities were affected by choices of morphological index (fractal dimension > sulcal depth > gyrification index > cortical thickness), brain parcellation (high-resolution > low-resolution), thresholding method (proportional > absolute), and network type (binarized > weighted). For the factor of similarity measure, its effects depended on the thresholding method used (absolute: Kullback–Leibler divergence > Jensen–Shannon divergence; proportional: Jensen–Shannon divergence > Kullback–Leibler divergence). Furthermore, longer data acquisition intervals and different scanner software versions significantly reduced the reliabilities. Finally, we showed that inter-site reliabilities were significantly lower than intra-site reliabilities for single-subject cerebral morphological networks. Altogether, our findings propose single-subject cerebral morphological networks as a promising approach for multicentric human connectome studies, and offer recommendations on how to determine analytical pipelines and scanning protocols for obtaining reliable results.

In this paper, an electro-hydrostatic actuator driven by dual axial-mounted magnetostrictive material rods-based pumps (MMPs) with a new type of active rectification valve is designed in the current study. Based on flow distribution of the active rectification valve and driving energy provided by two MMPs, the actuator can output continuous and bidirectional displacement. By establishing a mathematical model of the actuating system, using simulation techniques, the change rule of hydraulic cylinder’s motion state caused by different driving signals are studied and analyzed. Test equipment platform is constructed in the laboratory to test the output characteristics and confirm the feasibility of the new concept. The experimental results indicate that the maximum flow rate can reach approximately 2.7 L·min−1, while the operating frequency is 180 Hz.

Pilot two-stage proportional valves are widely used in high-power hydraulic systems. For the purpose of improving the dynamic performance, reliability, and digitization of the traditional proportional valve, a novel two-stage proportional valve with a pilot digital flow distribution is proposed from the viewpoint of the dual nozzle-flapper valve’s working principle. In particular, the dual nozzle-flapper is decoupled by two high-speed on/off valves (HSVs). First, the working principle and mathematical model of the proposed valve are determined. Then, the influences of the control parameters (duty cycle and switching frequency) and structural parameters (fixed orifice’s diameter and main valve’s spring) on the main valve’s motion are analyzed on the basis of theory, simulation, and experiment. In addition, in optimizing the value of the fixed orifice’s diameter, a new design criterion that considers the maximum pressure sensitivity, flow controllability, and flow linearization is proposed to improve the balance between the effective displacement and the displacement fluctuation of the main valve. The new scheme is verified by simulations and experiments. Experimental results of the closed-loop displacement tracking have demonstrated that the delay time of the main valve is always within 3.5 ms under different working conditions, and the tracking error can be significantly reduced using the higher switching frequency. The amplitude-frequency experiments indicate that a − 3 dB-frequency of the proposed valve can reach 9.5 Hz in the case of ±50% full scale and 15 Hz in the case of 0%–50% full scale. The values can be further improved by increasing the flow rate of the pilot HSV.

This article focuses on accurate identification of the critical moving characteristics of the high speed on/off valve. Typically, there are two strategies for identifying the critical moving characteristics, including calculation strategy based on force balance and strategy for detecting the coil current’s certain points. However, the accuracy of the two above strategies needs to be improved. Therefore, to improve the identification accuracy of the high speed on/off valve’s critical moving characteristics, an identification strategy for detecting the time derivative of the coil current is proposed. First, a mathematical model of the high speed on/off valve (including electromagnetic sub-model and mechanical-fluid sub-model) is established. And on this basis, relationship between the coil current’s derivative and the valve’s critical moving characteristics is analyzed which reveals the changing rule of the coil current’s derivative causing by the ball valve’s moving. Finally, the changing rule of the coil current’s derivative is verified by the comparative simulations and experiments, which also indicate that, with the proposed identification strategy, the maximum identification error of the critical opening/closing time is only within 6%, and the maximum identification error of the total opening/closing time is still small (2.9%), compared to other identification strategies in the previous literatures.

In this paper, the optimization is carried out for a synthetic jet based piezoelectric air pump. The structural parameters which affect the output flowrate are determined and orthogonal test method is used to find out the optimal values of these parameters. Driven by a 150 Vpp, 3.15 kHz sinusoidal voltage signal, the optimized pump reaches its maximum output flowrate of 2.79 L/min, which is 32.9% higher than that before optimization. Using the synthetic jet based piezoelectric air pump as gas source, an open-loop detection and closed-loop control system is built for electronic cooling. The open-loop experiments show that the heater’s surface temperature can be maintained at around 97.9 °C when the cooling distance is 60 mm, which is 32.5% lower than the initial value. The closed-loop control system can limit the temperature within the set interval. Both the open and closed-loop system show that the synthetic jet based piezoelectric air pump has application prospects in the field of electronic cooling.

Marine meteorology information is important for weather-sensitive marine activities while it is difficult to collect meteorological data in harsh environments. The objective of this paper is to design an unmanned surface vessel (USV) system, which consists of several subsystems, for marine meteorological data acquisition. Taking into consideration the reliability of the self-driving subsystem, a redundant autonomous driving module is designed to remove the broken controller. The stability and reliability of data transmissions are guaranteed by a multi-mode communication subsystem composed of radio and Beidou satellite communication modules. For the sake of the vessel safety, a navigation obstacle avoidance subsystem is employed in combination with vision sensors. The power subsystem including diesel generators and batteries is designed to extend the working time of meteorological monitoring. Moreover, an adaptive circular LOS guidance law is proposed to reduce the overshoot caused by the disturbance due to wind and wave. Finally, several experiments are conducted to validate the effectiveness of the system. The results show that the USV with designed system is able to sail across the tough marine area while showing important advantages for the acquisition and transmission of marine meteorological data in real time.

The calanoid copepod Acartia tonsa is a reference species in standardized ecotoxicology bioassay. Despite this interest, there is a lack of knowledge on molecular responses of A. tonsa to contaminants. We generated a de novo assembled transcriptome of A. tonsa exposed 4 days to 8.5 and 17 mg/L nickel nanoparticles (NiNPs), which have been shown to reduce egg hatching success and larval survival but had no effects on the adults. Aims of our study were to 1) improve the knowledge on the molecular responses of A. tonsa copepod and 2) increase the genomic resources of this copepod for further identification of potential biomarkers of NP exposure. The de novo assembled transcriptome of A. tonsa consisted of 53,619 unigenes, which were further annotated to nr, GO, KOG and KEGG databases. In particular, most unigenes were assigned to Metabolic and Cellular processes (34–45%) GO terms, and to Human disease (28%) and Organismal systems (23%) KEGG categories. Comparison among treatments showed that 373 unigenes were differentially expressed in A. tonsa exposed to NiNPs at 8.5 and 17 mg/L, with respect to control. Most of these genes were downregulated and took part in ribosome biogenesis, translation and protein turnover, thus suggesting that NiNPs could affect the copepod ribosome synthesis machinery and functioning. Overall, our study highlights the potential of toxicogenomic approach in gaining more mechanistic and functional information about the mode of action of emerging compounds on marine organisms, for biomarker discovering in crustaceans.

The large yellow croaker (Larimichthys crocea) has low hypoxia tolerance compared with other fish species, and the mRNA levels of hypoxia-inducible factor (HIF)-1α in its brain do not change markedly under hypoxic conditions. In this study, we investigated noncoding transcription in the hypoxic response mechanism of L. crocea. We generated a catalog of long noncoding RNAs (lncRNAs) from the brain of L. crocea individuals under hypoxic stress, investigated lncRNA expression patterns, and analyzed the HIF signaling pathway by RNA sequencing. Prolyl hydroxylase domain 2 (PHD2) expression significantly increased after 6 and 12 h of hypoxia, and a lncRNA (Linc_06633.1) was found in the upstream, antisense region of PHD2. Linc_06633.1 may be an important regulator that promotes PDH2 expression under hypoxia in L. crocea, and we constructed a regulatory profile of L. crocea under hypoxic conditions. To the best of our knowledge, it is the first study that has been conducted on hypoxia signaling pathway regulation by lncRNAs in L. crocea and elucidates the role played by lncRNAs in the regulation of the hypoxia stress response in teleost fish.

Interleukin-12, 16 and 34 are important pro-inflammatory cytokines, some of the most important components of the innate immunity system. Herein, we identified interleukin-12A (lcIL12A), 16 (lcIL16) and 34 (lcIL34) in large yellow croaker (Larimichthys crocea), and determined their expression profile in unchallenged and challenged tissues. The coding sequence (CDS) of lcIL12A comprised 600 bp long encoding a protein of 199 amino acids (aa), the CDS of lcIL16 was 2454 bp encoding a protein of 817 aa, and the CDS of lcIL34 was 657 bp encoding a protein of 267 aa. Phylogenetic analysis revealed similar results to homology comparison that lcIL12A was closest to IL12A of Dicentrarchus labrax (73%) and Serola dumerili (73%), while lcIL16 had the closest relation to Lates calcarofer (72.6%), and lcIL34 to Sparus aurata (88.9%). Multiple sequence alignment showed these interleukins were highly conserved with other vertebrate interleukins in their functional domains. Further, quantitative real time PCR (qPCR) analysis revealed that lcIL12A, lcIL16 and lcIL34 were constitutively expressed in all examined tissues, with significantly higher expression in spleen, liver and kidney. This was especially true for lcIL34 gene. Importantly, when challenged with polyinosinic:polycytidylic acid (poly I:C) and Vibrio anguillarum (V. anguillarum), the mRNA expressions of these interleukins were up-regulated in liver, spleen and kidney. Their top values got over 4 folds at least relative to their expression at time 0, and even lcIL12 reached 13.37 fold at 12-h point in spleen. These suggested their anti-viral and anti-bacterial roles and their involvement in the innate immune response of Larimichthys crocea. These results would have major implications in improving our understanding of the functions of interleukins to defend against pathogen infections in teleost species.

Interferon regulatory factor (IRF)-4, 8, and 9 are essential in host defense against pathogens. Here, the full-length coding sequence (CDS), protein structure, and immune response of IRF4/8/9 (lc IRF4/8/9) were characterized in large yellow croaker (Larimichthys crocea). The open reading frame of lcIRF4, lcIRF8 and lcIRF9 encoded putative proteins of 463,422 and 406 amino acids, respectively. These IRFs share high sequence homology with other vertebrate IRFs and were constitutively expressed in all examined tissues. IRFs were upregulated following stimulation with Vibrio anguillarum in the liver, spleen, and kidney. These results suggest that IRF4/8/9 are vital in the defense of L. crocea against bacterial infection and further increase our understanding of IRFs function in innate immunity in teleosts.

Microsatellite markers have been used for more than ten years to elucidate parentage relationships in aquaculture species. This study aimed to assess the feasibility of utilizing microsatellite markers for parentage determination in cuttlefish (Sepiella japonica) using simulations and real data analysis. We developed a panel of eight microsatellite markers in our lab. These markers were highly polymorphic with a mean of 10.1 alleles and an average expected heterozygosity value of 0.809. Using five simple sequence repeat markers, an allele frequency data-based simulation indicated that the combined exclusion probability values would be over 99%, whereas the rate of assignment success for the real data set was 91.8%. Mismatches caused by null alleles and scoring errors at microsatellite loci were the major reasons for the discrepancies between the simulations and real data analysis. We concluded that microsatellite markers can be used as a powerful tool to evaluate the effectiveness of enhancement and release programs for S. japonica.

The family Loliginidae is a diverse and economically valuable group of cephalopods. To investigate the evolution of their mitochondrial genomes, we characterized and compared these genomes among nine species, six newly sequenced. In all nine species, mitochondrial genomes followed the typical invertebrate canonical organization with 13 protein-coding genes, 22 tRNAs, and two rRNAs. However, different from the vertebrates, there were three non-coding control regions for most of the loliginid species. As for the gene arrangement and organization, the mitochondrial genome of Sepioteuthis lessoniana is totally different from the other eight species—there is only one control region, but 25 tRNAs. We analyzed the level of selective pressures imposed on different protein-coding genes, and found that stabilizing selection is strongest on cytb. We then tested the phylogenetic utility of this new mitochondrial genome data using 13 protein-coding genes and the results showed that Loliginidae is divided into two major lineages, with S. lessoniana sister to a clade comprising the remaining species. Loliolus beka, Loliolus japonica, Loliolus uyii, Uroteuthis chinensis, Uroteuthis edulis, Uroteuthis duvauceli, Heterololigo bleekeri, and Doryteuthis opalescens were recovered as a monophyletic group.

Gonadal-specific aromatase encoded by cyp19a1a is the important enzyme controlling estrogen biosynthesis in teleosts. In the present study, the cDNA sequence of cyp19a1a was cloned and characterized from miiuy croaker Miichthys miiuy. The cDNA encoded a protein of 519 amino acids with five structural regions. Higher identities of amino acid sequences and conserved structural regions were found between Mmcyp19a1a and other cyp19a1a genes. In addition, Mmcyp19a1a was clustered together with other seawater fishes. Immunohistochemical analysis revealed that Mmcyp19a1a was localized exclusively in the cytoplasmic of thecal and granulosa cells surrounding the oocytes. Both the protein and mRNA levels of Mmcyp19a1a were increased significantly at the stage III follicles (mid-vitellogenic) and then decreased along with vitellogenesis. Interestingly, strong immunoreactive signals were also detected in the supporting cells of connective tissues during ovarian development. A 1777bp promoter fragment of Mmcyp19a1a was also isolated, and functional analysis using an EGFP reporter fusion in zebrafish larvae presented positive signals in the above of yolk sac, where is the region of pronephros and germ plasm occur. The Mmcyp19a1a:EGFP expression pattern was generally consistent with the endogenous cyp19a1a genesis. These results indicate that the Mmcyp19a1a gene plays an important role during vitellogenesis and oocyte maturation. The constructor of Mmcyp19a1a:EGFP may provide a useful tool for genetic analysis of gonad development in teleost.

In natural environments, fish survive in polluted water by cadmium (Cd) throughout their whole life cycle. However, little information is available on Cd toxicity considering a life cycle assessment. The present study investigated effects of environmental levels of cadmium (0, 2.5, and 5μg/L) on immune responses in liver and spleen of zebrafish for 15 weeks, from embryos to sexually maturity. Nitric oxide (NO) levels and iNOS activity declined in liver and spleen of zebrafish exposed to 5μg/L Cd, suggesting an immunosuppressive effect. The result was further supported by the decreased transcriptional levels of proinflammatory cytokines by Cd, such as interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1β (IL-1β), and tumour necrosis factor-α (TNF-α) in liver. However, a sharp increase in the mRNA levels of these cytokines was observed in spleen of zebrafish exposed to Cd. The increased mRNA expression of these proinflammatory cytokines may be the secondary effect following immunosuppression and just reflect a compensatory mechanism for coping with the decreased immunity, which may explain an increase in mRNA levels and a decrease in iNOS activity in spleen of zebrafish exposed to Cd. In liver, the down-regulated mRNA levels of iNOS paralleled with the decreased iNOS activity, suggesting a synchronous response from a molecular level to a biochemical level. Positive correlations between mRNA expression levels of nuclear transcription factor κB (NF-κB) and proinflammatory cytokines were also observed, suggesting that NF-κB might be required for the protracted induction of inflammatory genes. The corresponding changes in the mRNA levels of the inhibitor of κBα (IκBαa and IκBαb) may form a feedback loop to restore transcriptional activity of NF-κB. Furthermore, splenic ROS levels were increased by 5μg/L Cd, possibly activating NF-κB pathway. Taken together, immunosuppressive effects and tissue-dependent compensatory responses were demonstrated in zebrafish after full life-cycle exposure to environmental levels of Cd, indicating a compromise between survival and immunity.

The aim of the present study was to evaluate the effects of abrupt salinity stress (12, 26 (control), and 40) on lipid peroxidation, activities and mRNA levels of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx, and GR), and gene expression of the Nrf2-Keap1 signaling molecules at different times (6, 12, 24, and 48 h) in the liver of large yellow croaker Pseudosciaena crocea. The results showed that lipid peroxidation was sharply reduced at 6 h and increased at 12 h before returning to control levels in the hypo-salinity group. Similarly, lipid peroxidation was significantly decreased at 6 h followed by a sharp increase towards the end of the exposure in the hyper-salinity group. Negative relationships between lipid peroxidation and antioxidant enzyme activities and positive relationships between activities and gene expression of antioxidant enzymes were observed, suggesting that the changes at molecular levels and enzyme activity levels may provide protective roles against damage from salinity stress. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for regulating these genes. Furthermore, there was a positive relationship between the mRNA levels of Nrf2 and Keap1, indicating that Keap1 plays an important role in switching off the Nrf2 response. In conclusion, this is the first study to elucidate effects of salinity stress on antioxidant responses in large yellow croaker through the Keap1-Nrf2 pathway.

Loliginidae includes many economically important species in trophic systems worldwide. Here, we investigated genetic relationships and diversity in this family. Sequence comparisons and phylogenetic analyses revealed considerable variations between mitochondrial 16 S rRNA gene and cytochrome coxidase subunit I gene among nine Loliginid species. We identified three similar non-coding regions in eight Loliginid species, but not in Sepioteuthislessoniana. We detected a single extended termination-associated sequence and three conserved sequence blocks among these eight species. Our results suggest that Loliginidae forms a major lineage, with S. lessoniana located at the most basal position and forming an individual clade as sister to the remaining species. Loligobeka, Loliolusjaponica, Loliolusuyii, Loligochinensis, Loligoedulis, Loligoduvauceli, Loligobleekeri, and Loligoopalescensare clustered into a monophyletic group. We identified repetitive elements and repeat numbers in the control regions.

We performed a phylogenetic estimation of the family Mytilidae in the East China Sea based on nuclear internal transcribed spacer (ITS) genes and two mitochondrial genes (COI and 16S RNA). Analysis of five mytilid species based on each of the three genes resulted in mostly congruent trees, although there were some discrepancies in the classification of these species. We combine the results obtained from the three separate analyses to provide a phylogenetic estimation of Mytilidae. We found that the Mytilidae was divided into two major lineages: in one clade, Mytilus galloprovincialis was grouped with Mytilus coruscus; in the second clade, Septifer bilocularis was placed at the basal position in an individual clade, and Perna viridis and Musculista senhousia were recovered as a monophyletic group. Although these finding provide important insights into the taxonomic relationships among the Mytilidae, many aspects of Mytilidae phylogeny remain unresolved. Further analysis based on more molecular information and extensive taxon sampling is necessary to elucidate the phylogenetic relationships among the major lineages within the Mytilidae.

Large yellow croaker is an important marine aquaculture species in China. The aim was to determine an appropriate protocol of artificial fertilization for family construction in the breeding programme based on two trials. In trial 1, luteinizing hormone-releasing hormone A3 (LHRHA3) was injected once, with a dosage of 2 μg/kg for females and 1 μg/kg for males. The latency time was in the range of 30–34 h. The maturation stage was checked by extracting a few eggs with a Pasteur pipette. The fertilization rate and hatching rate were 27 and 52%, respectively. The percentage of females with spawning difficulties was 30%. In trial 2, the females were injected LHRHA3 twice: with a first dose of 0.8 μg/kg and a second dose of 2 μg/kg, at an interval of 10 h, whereas the males were still injected once. The latency time was in the range of 29.5–35 h, determined by only observing courtship behaviour of males. The females with spawning difficulties decreased to 10%, and the fertilization rate and hatching rate also improved to 41 and 62%, respectively.

The wide use of ZnO nanoparticles in a number of products implies an increasing release into the marine environment, resulting in the need to evaluate the potential effects upon organisms, and particularly phytoplankton, being at the base of the throphic chain. To this aim, dose-response curves for the green alga Tetraselmis suecica and the diatom Phaeodactylum tricornutum derived from the exposure to nano ZnO (100 nm) were evaluated and compared with those obtained for bulk ZnO (200 nm) and ionic zinc. The toxic effects to both algae species were reported as no observable effect concentration (NOEC) of growth inhibition and as 1, 10, and 50% effect concentrations (EC1, EC10, and EC50). The toxicity decreased in the order nano ZnO > Zn2+ > bulk ZnO. EC50 values for nano ZnO were 3.91 [3.66-4.14] mg Zn/L towards the green microalgae and 1.09 [0.96-1.57] mg Zn/L towards the diatom, indicating a higher sensitivity of P. tricornutum. The observed diverse effects can be ascribed to the interaction occurring between different algae and ZnO particles. Due to algae motility, ZnO particles were intercepted in different phases of aggregation and sedimentation processes, while algae morphology and size can influence the level of entrapment by NP aggregates.This underlines the need to take into account the peculiarity of the biological system in the assessment of NP toxicity.

Up to date, little information is available on effects of circadian rhythm on metal-induced toxicity in fish. In this study, zebrafish were acutely exposed to 0.97mgL-1 cadmium for 12h either at ZT0 (the light intensity began to reached maximum) or at ZT12 (light intensity began to reached minimum) to evaluate the temporal sensitivity of oxidative stress and inflammatory responses in the brain of zebrafish. Profiles of responses of some genes at mRNA, protein and activity levels were different between ZT0 and ZT12 in the normal water. Exposure to Cd induced contrary antioxidant responses and similar inflammatory responses between ZT0 and ZT12. However, the number of inflammatory genes which were up-regulated was significantly greater at ZT12 than at ZT0. And, the up-regulated inflammatory genes were more responsive at ZT12 than at ZT0. At ZT12, antioxidant genes were down-regulated at mRNA, protein and activity levels. Contrarily, antioxidant genes were not affected at mRNA levels but activated at the protein and/or activity levels at ZT0. Reactive oxygen species (ROS) sharply increased and remained relatively stable when fish were exposed to Cd at ZT12 and ZT0, respectively. Positive correlations between ROS levels and mRNA levels of nuclear transcription factor κB (NF-κB) and between mRNA levels of NF-κB and its target genes were observed, suggesting that ROS may play an essential role in regulating the magnitude of inflammatory responses. Taken together, oxidative stress and immunotoxicity in the brain were more serious when fish were exposed to Cd in the evening than in the morning, highlighting the importance of circadian rhythm in Cd-induced neurotoxicity in fish.

The cuttlefish Sepiella japonica , was once one of the four major marine fisheries of China and has dramatically declined since the 1980s to near extinction. The artificial releasing project was launched for resource restocking since 2006. Aiming to provide some valuable information to the management of the releasing project, the mitochondrial DNA control region sequence was employed to assess the genetic variation of various geographical populations in present study. The genetic diversity of S. japonica was characterized with the low value of nucleotide diversity (0.00313 ± 0.070). Pairwise fixation index (Fst) analysis indicated insignificant genetic differentiation among southern populations (p > 0.05). However, the QD population showed the significant genetic differention with other populations (p

The study was carried out to evaluate the effects of low-dose zinc (Zn) pre-exposure on survival rate, new Zn accumulation, and mitochondrial bioenergetics in the liver and spleen of large yellow croaker exposed to high-dose Zn. To the end, fish were pre-exposed to 0 and 2 mg L−1 Zn for 48 h and post-exposed to 0 and 12 mg L−1 Zn for 48 h. Twelve milligrams Zn per liter exposure alone reduced survival rate, but the effect did not appear in the 2 mg L−1 Zn pre-exposure groups. Two milligrams per liter Zn pre-exposure also ameliorated 12 mg Zn L−1 induced new Zn accumulation, reactive oxygen species (ROS) levels, and mitochondrial swelling in the liver. However, these effects did not appear in the spleen. In the liver, 2 mg L−1 Zn pre-exposure apparently relieved 12 mg L−1 Zn induced down-regulation of activities of ATP synthase (F-ATPase), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH). The mRNA levels of these genes remained relatively stable in fish exposed to 12 mg L−1 Zn alone, but increased in fish exposed to 12 mg L−1 Zn with 2 mg L−1 Zn pre-treatment. In the spleen, 2 mg Zn L−1 pre-exposure did not mitigate the down-regulation of mRNA levels of genes and activities of relative enzymes induced by 12 mg L−1 Zn. In conclusion, our study demonstrated low-dose zinc pre-exposure ameliorated high-dose zinc induced mitochondrial dysfunction in the liver but not in the spleen of large yellow croaker, indicating an organ-specific effect.

Cadmium (Cd) is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem. The mechanisms underlying Cd-induced oxidative stress and immunotoxicity in fish remain largely unknown. In this study, adult female zebrafish were exposed to 0 (control), 1 mg L −1 Cd for 24 h and 96 h, and the oxidative stress and inflammatory responses induced by Cd were evaluated in the brain, liver and ovary. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) increased in a time-dependent manner after treatment with Cd in the brain and liver. The increase may result from the disturbance of genes including copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), inducible nitric oxide synthase (iNOS), and ciclooxigenase-2 (COX-2) at mRNA, protein and activity levels. Although ROS, NO and MDA were not significantly affected by Cd in the ovary, the up-regulation of Cu/Zn-SOD, CAT, iNOS, and COX-2 was observed. Exposure to Cd induced a sharp increase in the protein levels of tumor necrosis factor alpha (TNF-α) in the brain, liver and ovary, possibly contributing to activate inflammatory responses. Furthermore, we also found a dramatic increase in mRNA levels of NF-E2-related factor 2 (Nrf2) and nuclear transcription factor κB (NF-κB) at 24 h in the liver and ovary. The corresponding changes in the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1a and Keap1b) and the inhibitor of κBα (IκBαa and IκBαb) may contribute to regulate the transcriptional activity of Nrf2 and NF-κB, respectively. Contrarily, mRNA levels of Nrf2, NF-κB, Keap1, Keap1b, IκBαa and IκBαb remained stable at 24 and 96 h in the brain. Taken together, we demonstrated Cd-induced oxidative stress and immunotoxicity in fish, possibly through transcriptional regulation of Nrf2 and NF-κB and gene modifications at transcriptional, translational, post-translational levels, which would greatly extend our understanding on the Cd toxicity.

In order to save the communication resources and reduce the energy consumption of multiple Unmanned Surface Vessels (USVs), a finite-time consensus control algorithm based on event-triggered strategy is proposed. The distributed discontinuous control protocol is designed by the defined state error, and the trigger function for each USV is given. It is proved by Lyapunov theory that the finite-time heading consensus of multi-USVs can be achieved by choosing the appropriate control gain. Finally, numerical simulations are given to show the effectiveness of the proposed strategy. The multi-USV system only updates the controller when the trigger condition is reached, which reduces the network consumption, so it has important practical significance.

Chromodomain helicase/ATPase DNA-binding protein 1-like gene (CHD1L) is a new oncogene which has been confirmed to be crucial to the progression of many solid tumors. In the present study, the expression of CHD1L was found to be upregulated in intrahepatic cholangiocarcinoma (ICC), which was significantly associated with histological differentiation (P=0.011), vascular invasion (P=0.002), lymph node metastasis (P=0.008) and TNM stage (P=0.001). Kaplan-Meier survival analysis revealed that ICC patients with positive CHD1L expression had shorter overall and disease-free survival than those with negative CHD1L expression. Functional study found that CHD1L exhibited strong oncogenic roles, including increased cell growth by CCK-8 assay, colony formation by plate colony formation assay, G1/S transition by flow cytometry and tumor formation in nude mice. In addition, RNAi-mediated silencing of CHD1L inhibited ICC invasion and metastasis by wound healing, Transwell migration and Matrigel invasion assays in vitro and in vivo. Collectively, our results show that CHD1L is upregulated and promotes the proliferation and metastasis of ICC cells. CHD1L acts as an oncogene and may be a prognostic factor or therapeutic target for patients with ICC.

Chromodomain helicase/ATPase DNA-binding protein 1-like gene (CHD1L) is a new oncogene which has been confirmed to be crucial to the progression of many solid tumors. In the present study, the expression of CHD1L was found to be upregulated in intrahepatic cholangiocarcinoma (ICC), which was significantly associated with histological differentiation (P=0.011), vascular invasion (P=0.002), lymph node metastasis (P=0.008) and TNM stage (P=0.001). Kaplan-Meier survival analysis revealed that ICC patients with positive CHD1L expression had shorter overall and disease-free survival than those with negative CHD1L expression. Functional study found that CHD1L exhibited strong oncogenic roles, including increased cell growth by CCK-8 assay, colony formation by plate colony formation assay, G1/S transition by flow cytometry and tumor formation in nude mice. In addition, RNAi-mediated silencing of CHD1L inhibited ICC invasion and metastasis by wound healing, Transwell migration and Matrigel invasion assays in vitro and in vivo. Collectively, our results show that CHD1L is upregulated and promotes the proliferation and metastasis of ICC cells. CHD1L acts as an oncogene and may be a prognostic factor or therapeutic target for patients with ICC.

TAK1-binding proteins (TABs) are important immune protein involved in various intracellular signalling pathways. Here, TAB1-3 (lcTAB1-3) were characterized from Larimichthys crocea. The predicted 1524 bp coding sequence of lcTAB1 encoded a 507-residue protein, while lcTAB2 (2271 bp) and lcTAB3 (1836 bp) encoded 756 and 611 residue proteins, respectively. Their sequence shared conserved domain structures and functional sites with their orthologs from other species. The expression of lcTAB1-3 were detected in all tested tissues, which were upregulated in spleen, liver and kidney following Vibrio parahemolyticus infection. Immunofluorescence staining revealed that lcTAB1 were localized in cytoplasm, while lcTAB2 and lcTAB3 were in the endsome. Moreover, the NF-κB protein level was obviously upregulated after the co-overexpression of lcTAK1 and lcTABs, higher than that after the overexpression of lcTAK1 or lcTABs alone. Co-immunoprecipitation proved the direct interaction of lcTAB1/lcTAB2/lcTAB3 and lcTAK1. These findings indicated the roles of lcTABs in immune response of Larimichthys crocea.

The aim of the present study was to assess survival rate, Zn accumulation, reactive oxygen species (ROS) levels, oxidative damage and antioxidant responses after Zn exposure (2 and 8 mg L−1 Zn) at different exposure times (6, 12, 24, 48 and 96 h) in the liver of large yellow croaker. Survival rate was reduced at 96 h, and hepatic Zn content increased during 24–96 by 8 mg L−1 Zn. In the 2 mg L−1 Zn group, no fish died and the increase in Zn content merely occurred at 96 h. Exposure to 8 mg L−1 Zn induced accumulation of ROS, lipid peroxidation and protein carbonylation during the late stage of exposure. In contrast, exposure to 2 mg L−1 Zn did not result in oxidative damage, which may result from the up-regulation of antioxidant defenses. Although exposure to 8 mg L−1 Zn increased activities and mRNA levels of antioxidant enzymes during the early stage of exposure, including Cu/Zn–SOD, Mn–SOD, CAT, GPx and GR, the activities of these enzymes except Cu/Zn–SOD were inhibited at 96 h. Furthermore, a sharp increase in Nrf2 expression was observed in fish exposed to 8 mg L−1 at 6 and 12 h, and 2 mg L−1 at 12 h and 24 h, suggesting that Nrf2 was required for the protracted induction of these genes. The late increase in Keap1 expression may support its role in switching off the Nrf2 response. In conclusion, the present study demonstrated different effects of low- and high-dose waterborne Zn on antioxidant responses, which could contribute to the understanding of antioxidant and toxic roles of zinc on a molecular level.

In this study, we sequenced and characterized melanoma differentiation-associated antigen 5 (LcMDA5), laboratory of genetics and physiology 2 (LcLGP2) and mitochondrial antiviral signaling protein (LcMAVS) from large yellow croaker ( Larimichthys crocea ). The LcMDA5 encodes 969 amino acids and contains two caspase-associated and recruitment domains (CARDs), a DExDc (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal domain) and a C-terminal regulatory domain (RD). The LcLGP2 encodes 679 amino acids and contains a DExDc, a HELICc and a RD. The LcMAVS encodes 512 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif ( 269 PVQDT 273 ). Phylogenetic analyses showed that all the three genes of large yellow croaker are clustered together with their counterparts from other teleost fishes. The Real-time PCR analyses showed that all the three genes were found to be constitutively expressed in all examined tissues in large yellow croaker, but all with relatively low expression levels. Expression analyses showed that the three genes were all rapidly and significantly upregulated in vivo after poly (I:C) challenge in peripheral blood, liver, spleen and head kidney tissues. The results indicate that the LcMDA5, LcLGP2 and LcMAVS might play important roles in antiviral immune responses.

Gonadotropin-releasing hormone (GnRH) plays a vital role in the regulation of reproduction through interaction with a specific receptor (the GnRH receptor). In this study, the GnRH receptor gene from the cuttlefish Sepiella japonica (SjGnRHR) was identified and characterized. The cloned full-length SjGnRHR cDNA was 1,468 bp long and contained a 1,029 bp open reading frame encoding 342 amino acid residues, 8 bp of 5' untranslated regions (UTR), and 431 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 38.75 kDa and an isoelectric point of 9.47. In addition, this protein was identified as belonging to the rhodopsin-type (class A) G protein-coupled receptor family. The predicted amino acid sequence contained two N-linked glycosylation sites and 18 phosphorylation sites. Multiple sequence alignment, phylogenetic tree analysis, and three-dimensional structure modeling were conducted to clarify SjGnRHR bioinformatics characteristics. In vitro SjGnRHR expression was carried out using HEK293 cells and the pEGFP-N1 plasmid, to verify the transmembrane properties of this protein. The interaction between the S. japonica GnRH receptor and its ligand was clarified using internalization analysis. SjGnRHR transcriptional quantification confirmed the wide distribution of SjGnRHR in various S. japonica mature tissues. In addition, the transcriptional profile of SjGnRHR in the female brain and ovary during gonadal development was analyzed. Results indicate that GnRHR may be involved in diverse S. japonica physiological functions, especially in the control of reproduction.

To explore the effect of environment conditions on immune activity of fish, eight immune-associated genes responsible for innate immunity were selected from the GenBank, i.e. Pgrn-a, Ifit2, P-hepcidin, Lect2, β2m, Irf1, Il25 and Hsp96, and the mRNA expressions of them in the kidney of cultured large yellow croaker Larimichthys crocea in different sea areas in the East China Sea were examined with qPCR techniques. In the contrasts of immune-associated gene expression between areas and populations, significant differences were found, expression levels of these immune-associated genes were lower in the clear water area than in the poor water quantity area, and lower in May than in October. MY was more sensitive to environmental factors than DQ, which was coincident with the water quality in the culturing areas. Differential analyses of the expression levels of these immune-associated genes showed that significant up-regulation could be triggered by poor environmental factors. The expression patterns indicated that the expression levels of these genes were sensitive to ecological changes, thereby the immune-associated genes, especially Pgrn-a, Ifit2, β2m, Il25 and Hsp96, might serve as immediate and sensitive indicators of population immunologic vigor and ecosystem health. But the expression of immunity-associated genes at the level of gene transcription is highly influenced by multiple factors, and the exact causes or influencing factors of the up-regulation or down-regulation of these genes still need further thorough investigation.

The present study aimed to determine the effects of starvation on lipid content and antioxidant responses in the right and left lobes of liver in large yellow croaker. Fish were divided into three groups: the control fish fed normally and the fish starved for 4 and 12 days. The set of biomarkers were determined, including crude lipid and MDA contents, and mRNA levels and activities of copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). Starvation for 12 days decreased lipid content and increased MDA content and mRNA levels and activities of antioxidant enzyme genes tested in both lobes of liver. No significant difference in these biomarkers between both lobes of liver was observed in fish starved for 12 days. However, there were significant differences between both lobes of liver in lipid and MDA contents, activities of CAT and GR, and expression levels of Cu/Zn-SOD and GR in fish starved for 4 days. These observed differences between starved and fed fish and between both lobes of liver could be important biomarkers that contributed in separating starved from fed fish and short-term starved from long-term starved fish, respectively. Our study emphasized the same lobe of the liver should be sampled when evaluating biomarkers during starvation in fish.

AMP-activated protein kinase (AMPK) is a highly conserved and multi-functional protein kinase that plays important roles in both intracellular energy balance and cellular stress response. In the present study, molecular characterization, tissue distribution and gene expression levels of the AMPK α1 and α2 genes from turbot (Scophthalmus maximus) under salinity stress are described. The complete coding regions of the AMPK α1 and α2 genes were isolated from turbot through degenerate primers in combination with RACE using muscle cDNA. The complete coding regions of AMPK α1 (1722 bp) and α2 (1674 bp) encoded 573 and 557 amino acids peptides, respectively. Multiple alignments, structural analysis and phylogenetic tree construction indicated that S. maximus AMPK α1 and α2 shared a high amino acid identity with other species, especially fish. AMPK α1 and α2 genes could be detected in all tested tissues, indicating that they are constitutively expressed. Salinity challenges significantly altered the gene expression levels of AMPK α1 and α2 mRNA in a salinity- and time-dependent manners in S. maximus gill tissues, suggesting that AMPK α1 and α2 played important roles in mediating the salinity stress in S. maximus. The expression levels of AMPK α1 and α2 mRNA were a positive correlation with gill Na+, K+-ATPase activities. These findings will aid our understanding of the molecular mechanism of juvenile turbot in response to environmental salinity changes.

The complement system is an indispensable component in innate immunity. As one of them, complement component C6 plays an important role in the complement system, mediating membrane attack complex (MAC) formation and bacterial lysis. In this study, full-length complement component C6 (LycC6) cDNA (GenBank accession no. KT962992) was cloned from the large yellow croaker (Larimichthys crocea) and an ORF of 3156 bp was identified. The deduced amino acid sequence predicted that LycC6 contained conserved residues and domains known to be critical for C6 function. The quantitative real-time PCR analysis revealed that the LycC6 transcript was expressed in all the examined tissues. Complement component C3 is another central molecule in the complement system, converging the upstream complement signals and mediating the MAC assembly pathway formation. LycC6 was indispensable for active MAC formation after Vibrio anguillarum (V. anguillarum) challenge. Complement component C3 of large yellow croaker (LycC3) and LycC6 expression increased in the liver, spleen and kidney, and was the highest in the liver after V. anguillarum and polyinosinic:polycytidylic acid (Poly I:C) challenge. The results suggested that LycC6 might be an important immune-related gene, which is playing an important role in the immune defenses against bacterial infection. Statement of relevance The complement component C6 is an important component that plays a major role in the complement system. This study provides the first investigation of the cloning and characterization of the complement component C6 gene from Larimichthys crocea, and analysis of the expression of complement component C6 and complement component C3 of Larimichthys crocea after Vibrio anguillarum and acid Poly I:C challenge. The work described has not been published or is under consideration for publication elsewhere. The study was reviewed and approved by the Ethics Committee of Animal Experiments of Zhejiang Ocean University.