Soiny Mullet Research Articles

Effects of Microplastics on the Activity of Digestive-, Antioxidative- and Immune-related Enzymes in Soiny Mullet (Liza haematocheila Temminck & Schlegel, 1845) Larvae

Microplastics (MPs), novel pollutants in the water environment, can cause damage to fish, especially to fish larvae. However, the effects of MPs on fish in estuarine areas with high levels of MPs pollution are unknown. In this study, a 14-day exposure test was conducted to analyze the effects of MPs on soiny mullet larvae by measuring the activities of digestive enzymes, antioxidant enzymes, and immune enzymes in soiny mullet larvae after exposure to MPs of different particle sizes (0.5 μm, 0.1mg/L; 6 μm, 0.1mg/L).Results showed that MPs exposure significantly decreased the activities of pepsin (PPS), alpha-amylase (α-AMS), catalase (CAT), and superoxide dismutase (SOD), and significantly increased the activities of acid phosphatase (ACP) and alkaline phosphatase (ALP), among which the PPS, CAT, SOD, ACP, and ALP were more sensitive to exposure to small particle size MPs (0.5 μm).Overall, exposure to MPs reduced digestion, destroyed the antioxidant system, and triggered an immune response in soiny mullet larvae, especially the small particles of MPs (0.5 μm). The results provide basis for understanding the detriment of microplastic in fish.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila)

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

Structural analysis of toll-like receptor 18 from soiny mullet (Liza haematocheila): Giving insights on the ligand binding mechanism of fish specific TLRs

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of innate immune system, playing crucial roles in immune defense against pathogens. TLR18, a member of TLR1 family, is fish-specific TLR and involves in the immune response against bacterial infection. Currently, the structural biology of fish TLR18 is poorly elaborated. In this study, the structure and ligand binding of TLR18 (smTLR18) of soiny mullet (Liza haematocheila), an economically valuable aquaculture mugilid species, were analyzed. The extracellular domain (ECD) of smTLR18 formed an open-loop horseshoe-shaped structure with the concave surfaces made up of 19 parallel β-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel β-sheet (βA-βD) surrounded by 5 α-helices (αA-αE). Molecular docking analysis revealed that both ECD domain and TIR domain of smTLR18 could form homodimers. For the ECD homodimer, the main residues involved in dimer formation were located from LRR10 to LRR14. For the TIR homodimer, the residues involved in dimer formation were located in BB loop, αB helix, αC helix and DD loop. Ligand binding analyses revealed that peptidoglycans (PGNs) and lipopolysaccharides (LPS), two main bacterial pathogen-associated molecular patterns (PAMPs), were the potential ligands of smTLR18. The van der Waals and Coulombic interactions contributed to the interactions between smTLR18 and PGNs, while only van der Waals dominated the interactions between smTLR18 and LPS. The residues involved in ligands binding were located from LRR9 to LRR13. Our results provided the structural bases for elucidate the ligand binding of fish TLR18.

TLR13, TLR22, TRAF6, and TAK1 in the soiny mullet (Liza haematocheila): Molecular characterization and expression profiling analysis

Toll-like receptors (TLRs) and their associated signaling pathways play pivotal roles in the immune response to invading pathogens. Here, TLR13, TLR22, tumor necrosis factor receptor-associated factor 6 (TRAF6), and transforming growth factor-β-activated kinase1 (TAK1) were characterized in the soiny mullet (Liza haematocheila), representative mugilid species that is widely cultured in Asia. The four mullet genes, which shared characteristic features with their counterparts in other teleosts, were ubiquitously expressed in all of the examined tissues, albeit with different expression patterns. Following Streptococcus dysgalactiae infection, the four genes were upregulated to different degrees in various mullet tissues. These results indicated that the four genes were involved in the mullet immune response to bacterial infection. To the best of our knowledge, this is the first characterization of these four genes in mullet. Our results provide a basis for future studies of TLR signaling pathways in mullet, as well as for similar studies in other mugilids.

Antioxidant system of soiny mullet (Liza haematocheila) is responsive to dietary poly-β-hydroxybutyrate (PHB) supplementation based on immune-related enzyme activity and de novo transcriptome analysis

As a dietary supplement, poly-β-hydroxybutyrate (PHB) has been reported to positively influence growth, boost the immune system and enhance disease resistance in fish and shellfish. However, the protective mechanism is little known. Thus, the present study was conducted to evaluate the effect of PHB supplementation on immune-related enzyme activity and transcriptome-based gene expression in soiny mullet (Liza haematocheila). Results showed that dietary PHB supplementation could increase antioxidant enzyme activity, including total antioxidant capacity, catalase and superoxide dismutase. A total of 7,082,094,175 and 7,650,341,357 raw reads with mean length of 757 bp were obtained from control and PHB (dietary PHB supplementation at 2%) groups, respectively. There were 46,106 differentially expressed genes (DEGs) between control and PHB groups, including 21,828 upregulated and 24,278 downregulated DEGs. All the DEGs were classified into three gene ontology categories, and 312 DEGs related with immune system process and 760 with the response to a stimulus. Additionally, all DEGs were allocated to 261 Kyoto Encyclopedia of Gene and Genome pathways, and major immune-related pathways were detected, including MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. Moreover, the regulation of several observed immune-related genes was confirmed by qRT-PCR. Altogether, this study suggests that antioxidant system is more effective for dietary PHB supplementation and lays the foundation for further study on the precise immunostimulatory mechanism of PHB. Hopefully, it provides insights into exploring biomarker for assessment of immunostimulants in fish culture.

Direct cytotoxic activity of CD8+ T cells against Ichthyophthirius multifiliis in ginbuna crucian carp, Carassius auratus langsdorfii

The suppressor of cytokine signaling (SOCS) family members play crucial roles in regulating immune signal pathways by acting as inhibitors of cytokine receptor signaling. In this study, 10 SOCS genes were identified in soiny mullet (Liza haematocheila), an economically important aquaculture mugilid species in China and other Asian countries. Sequence comparison showed that the sequence identity between mullet SOCSs and their counterparts from other vertebrates ranged from 38.2% to 92.5%. All mullet SOCS genes were constitutively expressed in tissues examined, but their expression patterns were different. Further, following Streptococcus dysgalactiae infection, all mullet SOCS genes exhibited distinct expression patterns in tissues. These results suggest that SOCSs are involved in immune response to bacterial infection and provide the basis for understanding the complex cytokine regulatory network of teleosts.

Effects of dietary poly-β-hydroxybutyrate supplementation on the growth, immune response and intestinal microbiota of soiny mullet (Liza haematocheila)

Soiny mullet (Liza haematocheila) is an important economic fish species in China, but stress and diseases have seriously restricted its culture. There are no effective methods including vaccines to prevent or control these diseases. Alternative methods should be employed, such as using novel immunostimulant poly-β-hydroxybutyrate (PHB). The present study aimed to evaluate effects of dietary PHB supplementation on the growth, antioxidant enzymes activity, immune-related genes expression and intestinal microbiota in soiny mullet. The fish was fed for 30 or 60 days with six diets at different PHB supplementation of 0, 0.5, 1, 2, 4 and 8%, named as groups P0, P0.5, P1, P2, P4 and P8. The results showed that the weight gain and specific growth rate of fish in P2 and P0.5 groups were significantly higher than those in control P0 group at 30 and 60 days, respectively (P < 0.05). The antioxidant enzymes activity of catalase and superoxide dismutase in serum were significantly increased in P0.5/P1/P2 groups after 30 days. The transcriptional levels of penicillin-binding protein A and interleukin-8 analyzed by qRT-PCR were significantly upregulated in P2 and P4 groups compared to those in P0/P0.5/P1/P8 groups at 30 days. The transcriptional level of major histocompatibility complex class II in P2 group was significantly upregulated, and aldehyde oxidase downregulated compared to P0 group. Intestinal microbiota analysis by Illumina high-throughput sequencing showed that the microbiota diversity was not changed significantly, but the microbiota structure shifted significantly post PHB treatment. At the phyla level, Firmicutes and Proteobacteria were predominant in both P0 and P2 groups. At the genus level, the relative abundance of Bacillus spp. in P2 group increased significantly, and abundance of Achromobacter spp. decreased significantly. KEGG pathway analysis by PICRUSt showed that oral administration PHB significantly upregulated abundances of genes responsible for 10 pathways and downregulated genes involved in 17 pathways. In conclusion, soiny mullet fed with 2% PHB supplemental diets for 30 days showed better growth performance, higher antioxidant enzymes activity and immune-related genes expression. Their regulation of growth and immunity might be related with the intestinal microbiota change post PHB supplementation. It will provide very useful basic information to study the regulation mechanism of PHB in aquatic animals, and provide good green method to prevent disease in soiny mullet.

Identification and expression analysis of suppressors of cytokine signaling (SOCS) from soiny mullet (Liza haematocheila)

The suppressor of cytokine signaling (SOCS) family members play crucial roles in regulating immune signal pathways by acting as inhibitors of cytokine receptor signaling. In this study, 10 SOCS genes were identified in soiny mullet (Liza haematocheila), an economically important aquaculture mugilid species in China and other Asian countries. Sequence comparison showed that the sequence identity between mullet SOCSs and their counterparts from other vertebrates ranged from 38.2% to 92.5%. All mullet SOCS genes were constitutively expressed in tissues examined, but their expression patterns were different. Further, following Streptococcus dysgalactiae infection, all mullet SOCS genes exhibited distinct expression patterns in tissues. These results suggest that SOCSs are involved in immune response to bacterial infection and provide the basis for understanding the complex cytokine regulatory network of teleosts.

Cloning and Expression of β-Defensin from Soiny Mullet (Liza haematocheila), with Insights of its Antibacterial Mechanism.

Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the β-defensin (Lhβ-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhβ-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhβ-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) β-defensin. Realtime PCR revealed that Lhβ-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhβ-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhβ-defensin possessed a monomeric structure of a three-stranded anti-parallel β-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of β-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhβ-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel β1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two β1-strands. The most important interactions between β-defensin and membrane are the electrostatic interactions between Arg residues in β-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish β-defensins.

Transcriptome analysis of soiny mullet (Liza haematocheila) spleen in response to Streptococcus dysgalactiae.

Soiny mullet (Liza haematocheila) is becoming an economically important aquaculture mugilid species in China and other Asian countries. However, increasing incidences of bacterial pathogenic diseases has greatly hampered the production of the soiny mullet. Deeper understanding of the soiny mullet immune system and its related genes in response to bacterial infections are necessary for disease control in this species. In this study, the transcriptomic profile of spleen from soiny mullet challenged with Streptococcus dysgalactiae was analyzed by Illumina-based paired-end sequencing method. After assembly, 86,884 unique transcript fragments (unigenes) were assembled, with an average length of 991bp. Approximately 41,795 (48.1%) unigenes were annotated in the nr NCBI database and 57.9% of the unigenes were similar to that of the Nile tilapia. A total of 24,299 unigenes were categorized into three Gene Ontology (GO) categories (molecular function, cellular component and biological process), 13,570 unigenes into 25 functional Clusters of Orthologous Groups of proteins (COG) categories, and 30,547 unigenes were grouped into 258 known pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Following S.dysgalactiae infection, 11,461 differentially expressed unigenes were identified including 4658 up-regulated unigenes and 6803 down-regulated unigenes. Significant enrichment analysis of these differentially expressed unigenes identified major immune related pathways, including the Toll-like receptor, complement and coagulation cascades, T cell receptor signaling pathway and B cell receptor signaling pathway. In addition, 24,813 simple sequence repeats (SSRs) and 127,503 candidate single nucleotide polymorphisms (SNPs) were identified from the mullet spleen transcriptome. To this date, this study has globally analyzed the transcriptome profile from the spleen of L.haematocheila after S.dysgalactiae infection. Therefore, the results of our study contributes to better on the immune system and defense mechanisms of soiny mullet in response to bacterial infection, and provides valuable references for related studies in mugilidae species which currently lack genomic reference.

Molecular cloning, expression analysis and functional characterization of interleukin-22 in So-iny mullet, Liza haematocheila

In the present study, interleukin-22 (IL-22) from So-iny mullet (Liza haematocheila) was identified, and its tissue expression in both healthy and Streptococcus dysgalactiae-infected fish was examined. The full length cDNA sequence of mullet IL-22 was 1070bp, containing an open reading frame of 555bp. The deduced amino acid sequence shared high similarity (45.1–67.9%) with IL-22 from other fish species. Mullet IL-22 also contained an IL-10 family signature and four cysteine residues that were well conserved in other vertebrate IL-22 molecules. Mullet IL-22 mRNA was highly expressed in kidney, moderately expressed in liver and gut, and relatively weakly expressed in spleen, and its expression was significantly up-regulated in all the examined tissues following S. dysgalactiae infection. Furthermore, recombinant mullet IL-22 protein was shown to promote the expression of β-defensin in the four tissues and to increase the survival rate of the fish infected with S. dysgalactiae. Our results suggest mullet IL-22 plays an important role in the immune defense against bacterial infection and has the potential to be used to treat bacterial diseases in fish.

3-D modeling and molecular dynamics simulation of interleukin-22 from the So-iny mullet, Liza haematocheila

Background: Interleukin-22 (IL-22) plays an important role in the regulation of immune responses. However, little is known about its function or structure in fish. Results: The IL-22 gene was first cloned from So-iny mullet ( Liza haematocheila ), one of commercially important fish species in China. Then, 3-D structure model of the mullet IL-22 was constructed by comparative modeling method using human IL-22 (1M4R) as template, and a 5 ns molecular dynamics (MD) was studied. The open reading frame (ORF) of mullet IL-22 cDNA was 555 bp, encoding 184 amino acids. The mullet IL-22 shared higher identities with the other fish IL-22 homologs and possessed a conserved IL-10 signature motif at its C-terminal. The mullet IL-22 model possessed six conserved helix structure. PROCHECK, SAVES and Molprobity server analysis confirmed that this model threaded well with human IL-22. Strikingly, analysis with CastP, cons-PPISP server suggested that the cysteines in mullet IL-22 might not be involved in the forming of disulfide bond for structural stabilization, but related to protein-protein interactions. Conclusions: The structure of IL-22 in So-iny mullet ( Liza haematocheila ) was constructed using comparative modeling method which provide more information for studying the function of fish IL-22.

Comparison of Nekton Use for Cordgrass Spartina alterniflora and Bulrush Scirpus mariqueter Marshes in the Yangtze River Estuary, China

To test whether invasive Spartina alterniflora marshes were functionally equivalent to native Scirpus mariqueter marshes, the present study used bottomless lift nets (20 m2) during 12 high-tide events from August to October 2008 to compare nekton densities and biomass between the two marsh types in the Dongtan wetland. A total of eight species of fish, two species of shrimp, and three species of crab were collected. So-iny mullet Chelon haematocheilus, keeled mullet Liza carinata, Asian freshwater goby Acanthogobius ommaturus, and ridge-tail prawn Exopalaemon carinicauda dominated samples from the two marsh types and accounted for over 90% of the total catch. There were significantly greater densities and biomass (p 0.05) between the two marsh types were observed for densities and biomass of any species or total nekton in September and October 2008. Non-metric multidimensional scaling ordination did not show clear separation of samples between the two marsh types (r = 0.071, p = 0.159). Furthermore, there were no habitat-specific differences (p > 0.05) in the size distributions of the three numerically dominant species (C. haematocheilus, L. carinata, and A. ommaturus). We concluded that S. alterniflora marshes were utilized by nekton in a fashion similar to their utilization of native S. mariqueter marshes under similar physical conditions.

Stable isotopes in aquatic food web of an artificial lagoon in the Hangzhou Bay, China

Stable isotope values, δ13C and δ15N, were determined for four primary producers and 19 dominant consumers in a small artificial lagoon located in Hangzhou Bay. Based on these results the major pathways for energy flow and trophic structure of the artificial lagoon ecosystem were characterized. The mean δ13C values for the 19 consumers ranged from −22.99‰ to −14.24‰. Apart from so-iny mullet Liza haematocheila, the other 18 consumers had intermediate δ13C values between those of epibenthic microalgae and particulate organic matter (POM). The results of a multiple source linear mixing model (IsoSource model) indicated that 50% or more of the organic carbon in the tissues of most consumers was derived from epibenthic microalgae. This indicated that these primary producers were the main food source fueling the lagoon food web. The mean δ15N values for the 19 consumers varied between 4.93‰ and 12.97‰ and indicated four trophic levels in the lagoon. Four macroinvertebrates and zooplankton represented the primary consumers, whilst the other 14 consumers occupied the secondary and tertiary consumer levels. The 19 consumers were divided into three trophic guilds (detritivores/suspension feeders, omnivores and carnivores).