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To evaluate the effect of high voltage pulsed electric field (PEF) treatment (10-20 kV/cm, 5-15 min) on the structural characteristics and sensitization of crude extracts of arginine kinase from Fenneropenaeus chinensis. By simulated in vitro gastric juice digestion (SGF), intestinal juice digestion (SIF) and enzyme-linked immunosorbent assay (ELISA), AK sensitization was reduced by 42.5% when treated for 10 min at an electric field intensity of 15 kV/cm. After PEF treatment, the α-helix content decreased, and the α-helix content gradually changed to β-sheet and β-turn. Compared to the untreated group, the surface hydrophobicity increased and the sulfhydryl content decreased. SEM and AFM analyses showed that the treated sample surface formed a dense porous structure and increased roughness. The protein content, dielectric properties, and amino acid content of sample also changed significantly with the changes in the treatment conditions. Non-thermal PEF has potential applications in the development of hypoallergenic foods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca 2+ -inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca 2+ -independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca 2+ in invertebrates., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. Extensive research has shown that autophagy plays a pivotal role in both viral infection and replication processes. Despite the increasing research dedicated to autophagy, investigations into shrimp autophagy are relatively scarce., Results: Based on three different methods, a total of 20 members of the ATGs were identified from F. chinensis, all of which contained an autophagy domain. These genes were divided into 18 subfamilies based on their different C-terminal domains, and were found to be located on 16 chromosomes. Quantitative real-time PCR (qRT-PCR) results showed that ATG genes were extensively distributed in all the tested tissues, with the highest expression levels were detected in muscle and eyestalk. To clarify the comprehensive roles of ATG genes upon biotic and abiotic stresses, we examined their expression patterns. The expression levels of multiple ATGs showed an initial increase followed by a decrease, with the highest expression levels observed at 6 h and/or 24 h after WSSV injection. The expression levels of three genes (ATG1, ATG3, and ATG4B) gradually increased until 60 h after injection. Under low-salt conditions, 12 ATG genes were significantly induced, and their transcription abundance peaked at 96 h after treatment., Conclusions: These results suggested that ATG genes may have significant roles in responding to various environmental stressors. Overall, this study provides a thorough characterization and expression analysis of ATG genes in F. chinensis, laying a strong foundation for further functional studies and promising potential in innate immunity., (© 2024. The Author(s).)

Fenneropenaeus chinensis is a commercially important shrimp species cultured in China. This study investigated eight F. chinensis populations in China, including four geographical populations, three commercial breeds, and one wild population captured from the Yellow Sea. Population stratification analysis revealed that the Hebei geographical population and commercial breeding "Huanghai No. 4" were relatively independent and stable, reflecting a relatively closed breeding environment, whereas gene introgression was present between other populations. Selective signature analysis detected artificial selection for vision, growth, and disease resistance in the Hebei population. Neuronal development-related genes were detected to be under selection in the Changyi and Rizhao populations. Fertility of the Rizhao population was also investigated. Additionally, genes in the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathway were involved in the high pH tolerance of the "Huanghai No. 4" population. This study provided support for the genetic mechanism of parsing economic traits and the development of molecular breeding technologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fenneropenaeus chinensis is a migratory marine species with a suitable growth at 18-30°C. To prolong breeding season and reduce mortality in winter, breeding new shrimp varieties with cold tolerance is essential. Genes upregulated and highly expressed at low temperature are reasonable candidate genetic markers for the breeding of cold tolerant strain variants. This study screened genes with these features by comparing multiple low-vs. normal-temperature transcriptome groups. The results showed that nine genes were upregulated and highly expressed at low temperature in more than seven of the nine comparison groups. Six of them were identified as genes encoding transcription factor ATF2, RNA recognition motif domain-containing protein, cytochrome b5-like protein, troponin C, tubulin alpha-1, and 18S/5.8S/28S rRNA, respectively. Cold-inducible upregulations of ATF2, cytochrome b5, and rRNAs were novel findings in this study. The other three novel genes were predicted to encode a membrane-bound extracellular protein and two lncRNAs. Four of the screened genes were verified by real time RT-PCR, and their expression levels were consistent with the sequencing results, demonstrating the accuracy of the transcriptome sequencing data. Function analysis showed that ATF2 might be the master transcription factor regulating the expressions of proteins involved in cellular responses to cold. The other genes played a role in events such as enhancing translation, increasing energy, inhibiting apoptosis, and preserving cell integrity. The expression features of these nine genes suggested that they were of great significance to the cold tolerance of shrimp., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Protein phosphorylation and dephosphorylation are the most common and important regulatory mechanisms in signal transduction, which play a vital role in immune defense response. Our previous study has found the level of tyrosine phosphorylation was significantly changed in the hemocytes of Fenneropenaeus chinensis upon white spot syndrome virus (WSSV) infection. In order to explore the relationship between protein phosphorylation and WSSV infection, the quantitative phosphoproteomics was employed to identify differential phosphorylated proteins in hemocytes of F. chinensis before and after WSSV infection, and elucidate the role of key differential phosphorylated proteins in WSSV infection process. The results showed that a total of 147 differential phosphorylated proteins were identified in the hemocytes, including 64 phosphorylated proteins and 83 dephosphorylated proteins, which were mostly enriched in pyruvate metabolism, TCA cycle, glycolysis, and ribosomal biosynthesis. Functional analysis of differential phosphorylated proteins showed that they were involved in cell apoptosis, cell phagocytosis, cell metabolism and antiviral infection. A total of 236 differential phosphorylation sites were found, including 91 modified sites in the phosphorylation proteins and 145 modified sites in the dephosphorylation proteins. Motif analysis showed that these phosphorylation sites could activate mitogen-activated protein kinase, P70 S6 kinase and other kinases in hemocytes. Moveover, the phosphorylation levels of eukaryotic protein initiation factor 4E binding proteins and histone H3 were further determined by ELISA and Western blotting, which both exhibited a significant increase post WSSV infection and reach their peak levels at 6 and 12 h, respectively. Moreover, we found that lactate, a metabolite closely related to pyruvate metabolism, TCA cycle and glycolysis, was significantly increased in the hemocytes after WSSV infection. This study revealed the protein phosphorylation response in hemocytes of F. chinensis to WSSV infection, which help to clarify the response characteristics and virus resistance mechanism of hemocytes in F. chinensis, and also facilitate further understanding of the interaction between WSSV and shrimp hemocytes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Lower pH gives rise to a harmful stress to crustacean. Here, we analyzed the proteomic response of Fenneropenaeus chinensis from control pH (pH value 8.2) and low pH (pH value 6.5) - treated groups by employing absolute quantitation-based quantitative proteomic (iTRAQ) analysis. Among the identified proteins, a total of 76 proteins differed in their abundance levels, including 45 upregulated and 31 downregulated proteins. The up-regulation of proteins like citrate synthase, cytochrome c oxidase, V-type proton ATPase, glyceraldehyde-3-phosphate dehydrogenase and fructose 1,6-bisphosphate-aldolase as well as the enrichment of the DEPs in multiple metabolic processes and pathways illustrated that increased energy and substrates metabolism was essential for F. chinensis to counteract low pH stress. Ion transporting related proteins, such as Na + /K + /2Cl - cotransporter and calmodulin, participated in the homeostatic maintenance of pH in F. chinensis. There were significant downregulation expressions of lectin, lipopolysaccharide- and beta-1,3-glucan binding protein, chitinase, cathepsin L and beta-glucuronidase, which indicating the immune dysfunction of F. chinensis when exposure to low pH condition. These findings can extend our understanding on the defensive mechanisms of the low pH stress and accelerate the breeding process of low pH tolerance in F. chinensis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A number of white spot syndrome virus (WSSV)-binding proteins have been identified previously in the hemocytes of Fenneropenaeus chinensis. In order to further investigate the differential WSSV-binding proteins in hemocyte subpopulations, granular hemocytes and hyalinocytes were sorted from WSSV-infected shrimp by immunomagnetic bead (IMB) method. The results of ELISA and immuno-dot blot assay showed that the WSSV-binding activity of granular hemocytes proteins was much stronger than that of hyalinocytes proteins. And the percentage of WSSV-positive granular hemocytes was significantly higher than that of hyalinocytes post WSSV infection, indicating that granular hemocytes were more susceptible to WSSV infection. Moreover, a total of 9 WSSV-binding proteins were successfully identified in granular hemocytes and hyalinocytes by two-dimensional virus overlay protein binding assay (2D-VOPBA) and MALDI-TOF MS analysis, of which 3 binding proteins (arginine kinase, protease 1 and transglutaminase) existing in both hyalinocytes and granular hemocytes and 6 proteins (F 1 ATP synthase β-chain, hnRNPs, GAPDH, RACK1, β-actin and cellular retinoic acid) detected only in granular hemocytes. Among these identified WSSV-binding proteins, the transglutaminase (TG) was further recombinantly expressed, and the recombinant TG could be bound with WSSV. Subsequently, quantitative real-time PCR analysis showed that differential expression levels of WSSV-binding proteins were observed in granular hemocytes and hyalinocytes. The results of this study revealed that the WSSV-binding proteins were differentially expressed in granular hemocytes and hyalinocytes, which provided a deeper insight into the interaction between WSSV and hemocyte subpopulations., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

pH has a great impact on the distribution, growth, behavior, and physiology in many aquatic animals. The comparison of proteomics between normal and high pH stress samples was successfully achieved using iTRAQ proteomic analysis to screen key response proteins and pathways. After high pH stress, 124 upregulated and 41 downregulated proteins were identified. The higher expression levels of proteins like citrate synthase, glutathione S-transferase, glutathione peroxidase, and cytochrome c oxidase are associated with oxidative stress and mitochondrial dysfunction. The upregulation of glucose-regulated protein 78 indicated that the endoplasmic reticulum stress is induced by high pH stress. There were significant upregulation expressions of V-type H + -ATPase, Na + , K + -ATPase, 14-3-3 protein, as well as ATP-binding cassette transmembrane transporters after high pH exposure, which indicating their important roles in response to high pH stress. The abundance of proteins involved in protein glycosylation, oxidative pentose phosphate pathway, protein export, and glutathione metabolism were found enriched in high pH group than in control group. Quantitative proteomic profiling and integrated analysis with transcriptomic data provide new insights into the mechanisms underlying the molecular response to high pH stress in Fenneropenaeus chinensis.

The apoptosis of hemocytes plays an essential function in shrimp immune defense against pathogen invasions. In order to further elucidate the differential apoptotic responses of the granulocytes and the hyalinocytes in Fenneropenaeus chinensis post WSSV infection, the characteristics of apoptotic dynamics and viral proliferation in total hemocytes and hemocyte subpopulations were respectively investigated in the present work. The results showed that the apoptotic rate of hemocytes changed significantly, and the apoptosis-related genes also showed significantly differential expression responses during WSSV infection. Interestingly, we found that the apoptotic rate of virus-negative hemocytes was significantly higher than that of virus-positive hemocytes in the early stage of WSSV infection, while it was significantly lower than that of virus-positive cells in the middle and late infection stages. The difference of apoptosis between virus-positive and virus-negative hemocytes seems to be an important way for the WSSV to destroy the host's immune system and facilitate the virus spread at different infection stages. It was further found that the apoptosis rate of granulocytes was always significantly higher than that of hyalinocytes during WSSV infection, indicating that granulocytes have a stronger apoptotic response to WSSV infection. Moreover, a higher viral load was detected in granulocytes, and the density of granulocytes decreased more rapidly post WSSV infection, indicating that the granulocytes are more susceptible and vulnerable to WSSV infection compared with the hyalinocytes. These results collectively demonstrated that the apoptotic response in shrimp hemocytes was significantly influenced by the WSSV infection, and the differential apoptotic response of granulocytes and hyalinocytes to WSSV indicated the differences of antiviral mechanisms between the two hemocyte subpopulations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Cui, Liang, Tang, Xing, Sheng and Zhan.)

In our previous work, prohibitin1 (PHB1) was identified to be only expressed in granulocytes of Fenneropenaeus chinensis. In order to elucidate the potential immunological properties of prohibitins in hemocyte subpopulations, in this paper, the full-length cDNAs of PHB1 and PHB2 were firstly cloned from F. chinensis using rapid amplification of cDNA ends approach, and they were designated FcPHB1 and FcPHB2, respectively. Based on the sequence analysis and multiple sequence alignment, FcPHB1 and FcPHB2 were members of SPFH protein family. By quantitative real-time RT-PCR, the higher mRNA transcription levels of FcPHB1 and FcPHB2 were detected in intestine and hemocytes of F. chinensis, and these two genes in hemocytes were significantly up-regulated upon WSSV infection. The FcPHB1 and FcPHB2 were recombinantly expressed in Escherichia coli BL21 (DE3), and employed as immunogens to produce the polyclonal antibodies (PAbs) in rabbits. Indirect immunofluorescence assay (IFA) revealed that the FcPHB1 and FcPHB2 were located both in the cytoplasm and nuclei of hemocytes, which could also be specifically recognized by the PAbs against FcPHB1 or FcPHB2 in Western blot. Interestingly, it was found that FcPHB1 and FcPHB2 were only expressed in the granulocytes of heathy shrimp and highly expressed in the WSSV-infected granulocytes, however only weak expressions of FcPHB1 and FcPHB2 were observed in the hyalinocytes of WSSV-infected shrimp. Meanwhile, silencing of FcPHB1 and FcPHB2 genes were performed by small interfering RNA, and the results showed that the WSSV copies in hemocytes were increased by knockdown of either FcPHB1 or FcPHB2, and the cumulative mortalities of shrimp in the silenced groups were also markedly increased. These results demonstrated that FcPHB1 and FcPHB2 played important roles in anti-WSSV infection, and their differential expression characteristics in hemocyte subpopulations provided a further understanding of the immune functions of granulocytes and hyalinocytes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Myostatin (Mstn) inhibits muscle growth in vertebrates with endoskeleton, but it is still inconclusive that Mstn is a positive or negative regulator in crustacean with exoskeleton, and little information was available for its function on myogenesis. In this study, we identified and characterized the Mstn from Fenneropenaeus chinensis (FcMstn), and investigated its function on myogenesis and muscle growth. Two different cDNA sequences (2628 bp and 2604 bp) encoding for slightly different sizes of proteins were obtained for FcMstn, containing 86 bp of 5' untranslated regions (UTR) and 1258 bp of 3' UTR. The open reading frame of the long sequence and the short sequence contain 1284 bp and 1260 bp cDNA, encoding 427 and 419 amino acid sequence, respectively. Sequence analysis revealed that the overall protein sequence and specific functional sites of FcMstn were highly conserved with those in other crustacean species. In the early development stage, the muscle firstly appeared in nauplius stage and developed gradually until post larval, but the expression of FcMstn at mRNA and protein levels decreased from nauplius stage to post larval stage, indicating that Mstn involved in myogenesis as a negative regulator in shrimp. In the adult shrimp, the expression of FcMstn at mRNA and protein levels in muscle were significantly lower in the larger group than in the smaller group, and the diameter and number of muscle fiber of the muscle were significantly different between the two groups. Moreover, the shrimp with reduced level of FcMstn by RNAi displayed a dramatic faster growth rate compared with the control group. The present study demonstrates that FcMstn involved in myogenesis and muscle growth probably also as a negative regulator in shrimp like in vertebrates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metal pollution in Laizhou Bay along the Bohai Sea in China has been posing a risk on fishery species and hence may affect seafood quality. In this work, shrimps Fenneropenaeus chinensis were sampled from three sites, namely, a reference (site 6334) and two metal-polluted (sites 6262 and 7262) sites, located in Laizhou Bay. The metal concentrations in shrimp muscle tissues were tested using the ICP-MS technique. The Cr and Cu concentrations were the highest in the shrimp samples from site 7262, exceeding the national seafood safety standard Ⅱ, and the As concentration was much higher than the national seafood safety standard Ⅲ. NMR-based metabolomics indicated that metal pollution induced oxidative and immune stresses, damaged the muscular structure, and disrupted energy metabolism in shrimps at sites 6262 and 7262, in particular disturbed osmotic regulation in shrimps at site 7262. Glycine and serine could serve as biomarkers for Cd in F. chinensis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

pH has a great impact on the distribution, growth, behavior, and physiology in many aquatic animals. Here, we analyzed miRNA expression profiles of Chinese shrimp (Fenneropenaeus chinensis) from control pH (8.2) and low pH (6.5)-treated shrimp. Expression analysis identified 6 known miRNAs and 23 novel miRNAs with significantly different expression between control pH 8.2 and low pH 6.5; the predicted target genes of differentially expressed miRNAs were significantly enriched in organic acid metabolic process, oxidoreductase activity, coenzyme binding, cofactor binding, and collagen trimer. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including citrate cycle, pyruvate metabolism, cytokine-cytokine receptor interaction, tight junction, carbon metabolism, etc. Our survey expanded the number of known shrimp miRNAs and provided comprehensive information about miRNA response to low pH stress.

Raf is a member in the Ras/Raf/MAPKK/MAPK signaling transduction pathway. To obtain a better understanding of Raf in the interaction between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), the sequence of cDNA of Raf from F. chinensis (FcRaf) was obtained. The FcRaf gene contained a 2421 bp open reading frame (ORF). The FcRaf shared most characteristic of Raf protein, such as the Raf-like Ras-binding domain (RBD), phorbol esters/diacylglycerol binding domain (C1 domain), and catalytic domain of the serine/threonine kinases, Raf (STKc&#95;Raf). The sequence of functional domains of Raf protein was relatively conserved. The FcRaf mRNA was detected in the tissues of gill, muscle, and hepatopancreas from normal F. chinensis. The mRNA abundance level of FcRaf in the gill was the highest, which was 2.7-fold the level in the hepatopancreas. The expression level of FcRaf was significantly (P < 0.05) up-regulated in the tissues of gill, muscle, and hepatopancreas post WSSV-infection, which suggested that FcRaf might be involved in the interaction between F. chinensis and WSSV. Two SNP loci were identified in the ORF, one of which was a C-T mis-sense mutation, where an Ala was replaced by a Val, and induced the predicted protein secondary structure change. Considering the relatively low MAF (0.07), whether this mis-sense mutation was a detrimental mutation needs further investigation., (Copyright © 2018. Published by Elsevier Ltd.)

Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular response to inflammatory cytokines, environmental stress and pathogenic infection, However, compared with mammals and insects, the potential function of p38 MAPK from Chinese shrimp (Fenneropenaeus chinensis) in response to viruses and bacterial infection is limited. In the study, the immune responses of four genes MKK3, MKK4, p38 and ATF-2 from F. chinensis were investigated in defending against white spot syndrome virus (WSSV), Vibrio anguillarum (V. anguillarum) and Staphylococcus aureus (S. aureus) infection. Results demonstrated that the expression levels of these four genes were apparently modulated in hemocytes and gills when shrimp were stimulated by WSSV or bacteria, particularly at 3-24h after infection. MKK3, p38 and ATF-2 were most sensitive to V. anguillarum (Gram-negative bacteria), followed by WSSV and S. aureus (Gram-positive bacteria), while MKK4 gene was most sensitive to S. aureus, followed by WSSV and V. anguillarum. Knockdown of Fcp38 by RNA interference (RNAi) resulted in a reduction in the expression of FcMKK3 and FcATF-2. The results indicate that p38MAPK signaling pathway plays a role in defending against viral and bacterial infections in F. chinensis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In our previous work, granulocytes and hyalinocytes were successfully separated by immunomagnetic bead (IMB) method using monoclonal antibodies (mAbs) against granulocytes of shrimp (Fenneropenaeus chinensis). In order to elucidate the proteomic differentiation between granulocytes and hyalinocytes, in this paper, the differentially expressed proteins were analyzed between non-fixed/un-permeabilized (NFP) haemocytes and fixed/permeabilized (FP) haemocytes using two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS). Then the FP haemocytes were separated into two haemocyte subpopulations using IMB method, and the comparative proteome between granulocytes and hyalinocytes was investigated. The results showed that 10 differentially expressed protein spots were detected and identified as 4 proteins in the NFP haemocytes. Twenty one differentially expressed proteins were successfully identified between granulocytes and hyalinocytes, which include 4 unique expressed proteins in granulocytes, 4 significantly highly expressed proteins in granulocytes, and 13 significantly high expressed proteins in hyalinocytes. According to Gene Ontology annotation, the identified proteins between granulocytes and hyalinocytes were classified into six categories, including binding proteins, proteins involved in catalytic activity, enzyme regulator activity, structural molecule activity, translation regulator activity, and ungrouped proteins. Furthermore, quantitative PCR confirmed that the trend of transcription levels of three selected genes were consistent with the proteomic data from 2-DE. The results may lead to better understanding of the functions of haemocyte subpopulations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

MicroRNA (miRNA) is a class of small noncoding RNA, which is involved in the post-transcriptional regulation in all metazoan eukaryotes. MiRNAs might play an important role in the host response to virus infection. However, miRNAs in the aquatic crustacean species were not extensively investigated. To obtain a better understanding of the response of Chinese shrimp Fenneropenaeus chinensis to white spot syndrome virus (WSSV) infection, the sequence and expression profile of miRNAs in the hepatopancreas of WSSV-infected F. chinensis were obtained by the high-throughput Illumina HiSeq 2500 deep sequencing technique. A total number of 129 known miRNAs and 44 putative novel miRNAs were identified from the deep sequencing data. The peak size of miRNAs was 22 nt (37.0%). 25 miRNAs were significantly (P < 0.05) differentially expressed post WSSV infection. Six of the differentially expressed miRNAs were randomly selected for further verification by the real-time RT-PCR technique. The results showed that there was a consistency between the deep sequencing and real-time RT-PCR assay. The target genes of differentially expressed miRNAs were predicted. Each miRNA had 4 target genes on average. The results suggested that some specific miRNAs might be involved in the response of F. chinensis to WSSV infection, and further provided basic information for the investigation of specific miRNAs in F. chinensis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our previous study demonstrated that an integrin β subunit of Chinese shrimp ( Fenneropenaeus chinensis ) (FcβInt) plays an important role in white spot syndrome virus (WSSV) infection. In the present work, in order to further elucidate the potential role of FcβInt in WSSV infection, the recombinant extracellular domain of β integringene of F. Chinensis (rFcβInt-ER) was expressed in Escherichia coli BL21 (DE3), and the eukaryotic expression plasmid PcDNA3.1-FcβInt-ER (PFcβInt-ER) was also constructed. Far-western blotting was performed to determine the binding specificity of rFcβInt-ER to WSSV envelope proteins, and results showed that rFcβInt-ER was able to specifically interact with rVP31, rVP37, rVP110 and rVP187. Moreover, the blocking effects of mouse anti-rFcβint-ER antibodies were both detected in vivo and in vitro. The ELISA and Dot-blotting in vitro assays both showed that mouse anti-rFcβInt-ER antibodies could partially block the binding of WSSV to the hemocyte membrane of F. chinensis . In the in vivo assays, the mortality of shrimp injected with WSSV mixed with anti-rFcβInt-ER antibodies was delayed, and was lower than in the control group. While the shrimp were intramuscularly injected with PFcβInt-ER, transcripts of PFcβInt-ER could be detected in different shrimp tissues within 7 days, and the mortality of shrimp injected with PFcβInt-ER was also delayed and lower compared with the control group post WSSV challenge. Furthermore, gene silencing technology was also used to verify the effect of FcβInt in WSSV infection, and results showed that the expression levels of the WSSV immediate early gene iel , early gene wsv477 , and late gene VP28 and the mortality of F. Chinensis were all significantly decreased in the FcβInt knock-down hemocyctes compared to the control group. Taken together, these results suggest that FcβInt plays important roles in WSSV infection., Competing Interests: The authors declare no conflict of interest.

In the present study a cDNA encoding a phosphopyruvate hydratase (enolase) was cloned from the muscle of the Chinese shrimp (Fenneropenaeus chinensis) and named as FcEnolase. The cDNA of FcEnolase encoded a protein of 434 amino acid residues with a molecular mass 47.22 kDa. The residues 342-355 constituted the signature motif "LLLKVNQIGSVTES". A SNP locus (C96T) in the ORF at 96 bp was identified. The results showed that the FcEnolase was a conserved gene. In the normal F. chinensis, the mRNA level in the muscle was much higher (P < 0.05) than the mRNA level in the gill and hepatopancreas. To verify the mRNA level of FcEnolase in the F. chinensis post WSSV infection, a real-time RT-PCR was performed. In the WSSV-infected F. chinensis, the FcEnolase mRNA level was significantly (P < 0.05) up-regulated in the muscle at 12 and 24 h post challenge (hpc) to approximately 2.7-fold and 2.7-fold the mRNA level in the controls, respectively. The FcEnolase mRNA level in the gill was significantly (P < 0.05) down-regulated at 6 hpc to approximately 0.3-fold the mRNA level in the control, followed by a significant (P < 0.05) up-regulation at 12 hpc to approximately 2.8-fold the mRNA level in the control. There was no obvious change of FcEnolase mRNA level in the hepatopancreas during the infection process. The expression profile coincided with the fact that WSSV primarily infects the tissues of muscle and gill, but hardly infects hepatopancreas. To verify the protein level of FcEnolase post WSSV infection, a Western blot was performed. The FcEnolase protein level in the muscle at 24 hpc significantly (P < 0.05) increased to approximately 2.1-fold the level in the control. These results showed the characterization of FcEnolase and suggested that the FcEnolase might be involved in the response of F. chinensis to WSSV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We investigated the antioxidant activity of taurine rich water extract from the cephalothorax of Fenneropenaeus chinensis (FCC). The antioxidant potency of water extract from FCC was assessed using various assay methods, such as DPPH (1,1-diphenyl-2-picrylhydrazyl), alkyl radical scavenging activity, ABTS (2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonic acid ammonium salt)) radical scavenging activity and Ferric reducing antioxidant power (FRAP) assay. The DPPH and alkyl radical scavenging activities of FCC were dose-dependently increased. The lipid peroxidation was estimated using ferric thiocyanate (FTC) assay and thiobarbituric acid (TBA) methods. However, a higher lipid peroxidation activity was observed in TBA method than FTC method. The results of the present study suggested that the FCC extract potentially scavenged the free radical and reduced oxidative stress. Therefore, the present study is concluded that the FCC extract could be a potential source of antioxidant activity.

In our previous work, two monoclonal antibodies (Mabs) against granulocytes of shrimp (Fenneropenaeus chinensis) had been produced, in this paper, haemocyte subpopulations were analyzed by flow cytometry (FCM) using the Mabs. Then immunomagnetic bead (IMB) method was applied for separation hyalinocytes and granulocytes using the Mabs. The separated hyalinocytes and granulocytes were analyzed by FCM, indirect immunofluorescence assay, Giemsa staining and transmission electron microscopy, respectively. The results showed the proportion of hyalinocytes in haemolymph of F. chinensis was 15.14 ± 1.22%, and that of granulocytes was 75.43 ± 2.31%. After two times separation by IMB, the purity rate of hyalinocytes and granulocytes was 96.27 ± 1.06% and 98.13 ± 0.86%, respectively. The hyalinocytes possessed 0.60-0.85 in nucleus/cytoplasm (N/C) ratio and had few granule in cytoplasm, whereas the separated granulocytes with N/C ratio of 0.12-0.36 and high electronic density of double membrane granules. The results reported the separation of haemocyte subpopulations using Mabs in shrimp for the first time, and the hyalinocytes and granulocytes isolated by IMB could be used for their differential protein analysis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mitogen-activated kinase kinase (MAPKK) is an important gene involved in the host-virus interaction process. To obtain a better understanding of MAPKK in the interaction process between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), we cloned the sequence of an MAPKK cDNA from F. chinensis (FcMAPKK) and investigated the effect of FcMAPKK on WSSV infection. The results showed that the FcMAPKK gene contained a 1227 bp open reading frame (ORF), which encoded a highly conserved protein with a serine/threonine protein kinase catalytic (S&#95;TKc) domain. The deduced amino acid sequence of FcMAPKK shared identities between 11.9 and 92.6% with MAPKKs from vertebrate, invertebrate, plant and fungus species. The FcMAPKK was expressed in all the examined tissues in the normal F. chinensis. FcMAPKK expression level was highest in the hepatopancreas where it was approximately 2.6-fold the expression level in the gill, and lowest in the muscle where it was approximately 0.3-fold the expression level in the hepatopancreas. The FcMAPKK expression levels in the muscle, gill, and hepatopancreas were all changed post WSSV challenge. The FcMAPKK expression was significantly (P < 0.01) up-regulated in the muscle of F. chinensis at 48 h post WSSV infection. The WSSV began to replicate quickly in the normal F. chinensis at 48 h post infection, while the WSSV replication in the U0126-treated F. chinensis could be significantly (P < 0.05) inhibited. The results suggested that FcMAPKK might be involved in the WSSV infection process, and hijacking of FcMAPKK might be required for WSSV replication in F. chinensis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

As the direct executors of biological function, the expression level of proteins will reveal the molecular mechanisms regulating WSSV acute infection more directly. In the present study, the iTRAQ approach was applied to identifying differentially expressed proteins in Chinese shrimp during WSSV latent infection and acute infection. A total of 4051 unique peptides corresponding to 1286 proteins were identified. 118 unique proteins showed differential up-regulation and 122 proteins were down-regulated in shrimp during WSSV acute infection compared with those in WSSV latent infection stage. A number of proteins related to actin-myosin cytoskeleton process, including myosin, actin, tubulin, clathrin, and tropomyosin were found up-regulated in shrimp at WSSV AI stage, indicating that the phagocytosis process was involved in WSSV AI stage. The apoptosis process in shrimp during WSSV AI seemed to be inhibited because some proteins suppressive on apoptosis were up-regulated, such as ALG-2 interacting protein x, Hsp90, 14-3-3-like protein, peroxiredoxin 5, peroxiredoxin 6 and adenine nucleotide translocase 2. Association analysis between the proteomic data and the previous transcriptome data was performed. Quantitative real-time PCR and western blot were carried out to verify the reliability of the proteomics data. The present study provided a comprehensive view of molecular mechanisms regulating WSSV acute infection at the protein level., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Haemocyanin (Hc) is frequently reported to vary significantly by physiological status and environmental stress in Crustaceans. In this paper, the shrimp Fenneropenaeus chinensis was infected with different concentrations of white spot syndrome virus (WSSV) and Vibrio harveyi. Then, the variation of Hc and total protein content of the haemolymph (TPCH) were investigated using the established double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and Coomassie brilliant blue method, respectively. The results showed that the Hc content peaked at 12 h post-infection (PI) in the 10(-2), 10(-4) and 10(-6) viral supernatant (VS) groups, and the maximum was 93.03 ± 2.55 mg ml(-1), 77.57 ± 6.02 mg ml(-1) and 70.25 ± 3.96 mg ml(-1), respectively. TPCH reached the maximum of 108.18 ± 1.36 mg ml(-1) and 103.49 ± 1.33 mg ml(-1) at 12 h PI in the 10(-2) and 10(-4) VS groups, respectively. The maximum was 96.94 ± 1.06 mg ml(-1) at 24 h PI in the 10(-6) VS group. In the V. harveyi infection groups, the Hc content reached a maximum of 87.97 ± 4.39 mg ml(-1) at 36 h PI in the 10(6) CFU ml(-1) group, 73.74 ± 4.38 mg ml(-1) and 72.47 ± 2.09 mg ml(-1) at 12 h PI in the 10(7) and 10(8) CFU ml(-1) groups, respectively. TPCH reached a maximum of 111.16 ± 0.86 mg ml(-1) at 36 h PI in the 10(6) CFU ml(-1) group, 100.41 ± 0.51 mg ml(-1) and 101.94 ± 0.47 mg ml(-1) at 12 h PI in the 10(7) and 10(8) CFU ml(-1) groups, respectively. These data showed that both Hc content and TPCH varied as the same extent after infection. The up-regulation of the Hc content at 6-36 h PI might be a reference threshold for shrimp infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To investigate the role of cytoplasmic dynein in white spot syndrome virus (WSSV) infection, the full-length cDNA of cytoplasmic dynein intermediate chain (FcDYNCI) was cloned in Fenneropenaeus chinensis, which consists of 2582 bp and encodes a polypeptide of 660 amino acids. Sequence analysis and multiple sequence alignment displayed that FcDYNCI was a member of cytoplasmic dynein 1 family. The FcDYNCI mRNA was most highly expressed in hemocytes, which was significantly up-regulated post WSSV infection. At 12 h post infection (hpi), confocal microscopic observation showed that WSSV could be co-localized with cytoplasmic dynein in hemocytes. After silencing by specific FcDYNCI dsRNA, the FcDYNCI mRNA level and the protein amount of FcDYNCI in hemocytes both exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited the greatest decreases at 24 hpi. These results suggested that cytoplasmic dynein was involved in WSSV infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Current knowledge on cis-regulatory elements of immune genes of shrimp is poor. In this study, the genomic sequence of the Fenneropenaeus chinensis anti-lipopolysaccharide factor (ALFFc) gene was obtained by using PCR and genome walking techniques, and the promoter was identified. The ALFFc gene contained three exons interrupted by two introns. Immune-related transcription factor binding sites recognized by nuclear factor-kappa B, octamer binding protein 1, GATA binding factor 1 and specificity protein 1 were identified in the regin from +1 to -702. The activity of ALFFc promoter was analyzed in insect sf9 cell lines. The putative promoter sequence of pALF-702 drive the expression of reporter EGFP gene successfully by adding lipopolysaccharide or (1,3)-β-D-glucan, but the shorter promoter sequence pALF-318 is only by (1,3)-β-D-glucan. The results pointed out that these transcription elements might contribute to the differences in promoter of ALFFc. Our results would provide supports for future studies to identify the functional transcription elements in the ALF promoter and to expand our knowledge on regulation of innate immune genes in Chinese shrimp., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Extracellular signal-regulated kinase (ERK) is a serine/threonine-specific protein kinase, which participates in signaling transduction pathways that control intracellular events, including resumption of meiosis, embryogenesis, cell differentiation, cell proliferation, cell death and response to radiation. Some virus species evolved the ability to hijack the host cell ERK signaling transduction pathway for viral replications and gene expressions. To obtain a better understanding of ERK, we cloned a cDNA encoding ERK from the muscle of Fenneropenaeus chinensis (FcERK). The FcERK contained a 1098 bp open reading frame (ORF) encoding a protein of 365 amino acid residues with a conserved phosphorylation motif TEY in the kinase activation loop. Pair-wise and multiple sequence alignment revealed that ERK is highly conserved across taxa. The FcERK gene expressions in the hepatopancreas and gill were noticeably higher than the expression observed in the muscle. A challenge test was performed to reveal the responses of FcERK in different tissues to white spot syndrome virus (WSSV) infection. Post WSSV challenge, the FcERK expression in the gill significantly increased during the early stage of the viral infection, the FcERK expression in the muscle increased later than that in the gill, and the FcERK expression in the hepatopancreas significantly decreased. The FcERK gene expression profile accorded with the results that the virus primarily infects tissues originating from the ectoderm, with less infection of the tissues originating from the mesoderm, and hardly any infection in the tissues originating from the entoderm. Two single nucleotide polymorphisms (SNPs) were identified in the FcERK gene, involving C/T transition. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps were obtained using a high-resolution melting (HRM) method. In the two groups, the MAFs of both sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distributions of both mutation sites between the two groups were not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for disease control., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cathepsin B is a unique member of the cathepsin superfamily, which acts as both an endopeptidase and peptidyl-dipeptidase. To obtain a better understanding of this enzyme, we cloned a cDNA encoding cathepsin B from the muscle of Fenneropenaeus chinensis (FcCB). FcCB contained a 996-bp open reading frame (ORF) encoding a protein of 331 amino acid residues with a putative signal peptide and a propeptide&#95;C1 at the N-terminal, a glutamine oxyanion hole and active site cysteine, histidine and asparagine residues. A region from residue 79 to 327 conferred the peptidase activity of FcCB. Pair-wise and multiple sequence alignment with 17 other organisms, including ten different vertebrate species, five different invertebrate species and two different plant species, indicated that the signal peptide and the propeptide&#95;C1 at the N-terminal of FcCB were less conserved than the mature protein, except when compared with Penaeus monodon, Litopenaeus vannamei and Marsupenaeus japonicas, all of which belong to the genus Penaeus. The expression of FcCB in the hepatopancreas was higher than that in the gill. The expression of FcCB in the gill was higher than that in the muscle. A challenge test was performed to reveal the responses of FcCB in different tissues to white spot syndrome virus (WSSV) infection, which causes serious economic losses in the shrimp farming industry. The FcCB gene expressions in the ectoderm, mesoderm and entoderm were not the same prior to WSSV infection, but at 6 h after WSSV challenge, the FcCB expression in the gill, hepatopancreas and muscle was up-regulated, suggesting that FcCB might be involved in the immune response to WSSV. Three single nucleotide polymorphisms (SNPs) were identified in the FcCB gene, involving C/T transitions, which are known as mutation hot spots. Notably, the three SNPs constituted a haplotype that can be used as an indicator of the haplotype block. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps, were obtained using a high-resolution melting (HRM) method. Associated factors, including observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF) and P-values for the deviation from Hardy-Weinberg equilibrium (HWE), were obtained. For the association analysis with WSSV resistance, the P-values were calculated using Pearson's chi-square test. In the two groups, the MAFs of all sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distribution of the C-984T mutation site between the two groups was not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for solving the WSSV problem., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rac1 is a ubiquitous GTP-binding protein that plays a crucial role in multiple cellular processes. In the present study, a Rac1 homolog (FcRac1) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The open reading frame (ORF) of FcRac1 consists of 579 bp encoding 192 aa. The predicted molecular weight (MW) of the deduced amino acid sequence of FcRac1 was 21.46 kDa, and its theoretical pI was 8.62. Homology analysis showed that the amino acid sequence of Rac1 had high conservation among those from different species. Phylogenetic analysis showed that FcRac1 closely related to Rac1 proteins from other arthropods. FcRac1 showed the highest expression level in the hemocytes. In situ hybridization detection showed that it distributed in all types of hemocytes. Recombinant protein of FcRac1 showed apparent activity of GTPase. The transcription of FcRac1 in juvenile shrimp changed after bacteria or WSSV challenge. The present data suggests that FcRac1 might play important roles in the innate immunity of shrimp., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44-53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNA was expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bioflocs contribute to the nutrition and growth of some penaeid shrimp, such as L. vannamei, by providing an additional food source in intensive, limited exchange production systems. However, it remains unclear whether other penaeids could use bioflocs as food sources. Furthermore, little is known concerning the feeding mechanism of shrimp for harvesting and consuming bioflocs. This study determines whether three penaeid species could use bioflocs as food sources and suggests mechanical functions of the third maxilliped to collect bioflocs in a water column. The postlarvae of L. vannamei, F. chinensis, and M. japonicus were assessed in 14-day culture trials with four treatments: biofloc water with feeding (BF-f), natural seawater with feeding (SW-f), biofloc water without feeding (BF-nf), and natural seawater without feeding (SW-nf). Litopenaeus vannamei exhibited a significantly higher specific growth rate (SGR) in the BF-f treatment than in the SW-f treatment, whereas the other two species did not demonstrate any differences in their SGR and survival rate between BF-f and SW-f treatments, suggesting that bioflocs might contribute to the growth of L. vannamei postlarvae but do not affect the survival and growth of F. chinensis or M. japonicus. The results obtained from the non-feeding experiments implied a filter feeding habit of L. vannamei and a cannibalistic habit of F. chinensis. Differences in the third maxillipeds are relevant in this regard. The feeding mechanism of M. japonicus with intermediate features of setae requires further study. The present study may contribute to understanding the different feeding abilities of bioflocs as food source in currently farmed penaeid shrimp in the Far East Asian countries. [ABSTRACT FROM AUTHOR]

Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5 and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235 bp (28.0%), 307 bp (36.6%), 138 bp (16.5%) and 158 bp (18.9%), respectively. Furthermore, 556 bp fragment of the sequence was used to discuss the phylogenetic relationship among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus. [ABSTRACT FROM AUTHOR]