Ecytonucleospora hepatopenaei n. gen. et comb. (Microsporidia: Enterocytozoonidae): A redescription of the Enterocytozoon hepatopenaei (Tourtip et al., 2009), a microsporidian infecting the widely cultivated shrimp Penaeus vannamei.

The microsporidian Enterocytozoon hepatopenaei from Penaeus vannamei (EHPPv) was redescribed on the basis of spore morphology, life cycle, pathology, and molecular character. Compared with the Enterocytozoon hepatopenaei isolated from Penaeus monodon (EHPPm), described by Tourtip et al. in 2009, new features were found in EHPPv. Electron microscopy demonstrated that EHPPv was closely associated with the nucleus of host cell. The merogony and sporogony phages were in direct contact with the cytoplasm of host cells, whereas some of the sporoblasts and the spores were surrounded by the interfacial envelope. Mature spores of EHPPv were oval and monokaryotic, measuring 1.65 ± 0.15 µm × 0.92 ± 0.05 µm. Spores possessed many polyribosomes around a bipartite polaroplast and the polar filament with 4-5 coils in two rows. Phylogenetic analyses showed all Enterocytozoon hepatopenaei isolates shared a common ancestor. Based on the morphological and molecular analyses, we propose the establishment of a new genus Ecytonucleospora and transferring Enterocytozoon hepatopenaei to the genus Ecytonucleospora, retaining the specific epithet hepatopenaei that Tourtip et al. proposed in recognition of their first research, as the new combination Ecytonucleospora hepatopenaei n. comb. Furthermore, it was suggested Enterospora nucleophila, Enterocytozoon sp. isolate RA19015_21, and Enterocytozoon schreckii be assigned into this new genus.

PvML1 suppresses bacterial infection by recognizing LPS and regulating AMP expression in shrimp.

Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of Drosophila and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named PvML1 was characterized in Penaeus vannamei. We found that PvML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. PvML1 was highly expressed in hemocytes and remarkably upregulated after Vibrio parahemolyticus challenge. Furthermore, the binding and agglutinating assays showed that PvML1 possessed strong binding activities to LPS and its key portion lipid A as well as Vibrio cells, and the binding of PvML1 with bacterial cells led to the agglutination of bacteria, suggesting PvML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating V. parahemolyticus with recombinant PvML1 promoted bacterial clearance in vivo and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of PvML1 remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of PvML1 severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by PvML1 with the AMP expression. Interestingly, we found that PvML1 interacted with the extracellular region of PvToll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by PvML1 might be described as follows. PvML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated PvToll2-mediated signaling pathway by interacting with PvToll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain.

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Newly identified type II crustin (SpCrus2) in Scylla paramamosain contains a distinct cysteine distribution pattern exhibiting broad antimicrobial activity.

Type II crustins are the most abundant type of crustins in shrimps that exhibit remarkable sequence diversities and broad antibacterial activities. This study characterized a novel type II crustin, SpCrus2, in the mud crab Scylla paramamosain. The SpCrus2 cDNA sequence is 620-bp long with a 495-bp open reading frame encoding a 164-amino acid protein. In the deduced protein, a 17-amino acid signal peptide, a glycine-rich hydrophobic region (GRR), and a cysteine-rich region (CRR) containing a whey acidic protein domain were predicted. SpCrus2 shares high similarity with most type II crustins (types IIa and IIb crustins) in shrimps but has a novel distribution pattern of cysteine residues that is distinct from most crustins. SpCrus2 and PlCrus3 from Pacifastacus leniusculus share high similarity and the same distribution pattern of cysteine residues. Thus, we proposed them as type IIc crustins. SpCrus2 is mainly distributed in the gills and can be up-regulated through Vibrio parahemolyticus or Staphylococcus aureus challenge. To investigate the biological functions of SpCrus2 and the underlying mechanisms, SpCrus2, GRR, CRR, and the mutant of CRR (CRR-M, the cysteine distribution pattern is mutated into that in most conventional crustins) were all overexpressed and purified. SpCrus2 GRR itself, as a glycine-rich amphiphilic peptide, exhibited evident antibacterial ability against Gram-negative bacteria, whereas CRR possessed potent antibacterial activity against Gram-positive bacteria. Either GRR or CRR exhibited weaker antibacterial activity than the whole protein of SpCrus2, indicating that GRR and CRR synergized to exert their potential antibacterial functions. In addition, CRR exhibited slightly stronger antimicrobial activity than CRR-M, suggesting that SpCrus2 containing this novel cysteine distribution pattern may exhibit stronger antimicrobial activity than most type II crustins with the conventional distribution pattern of cysteine residues. The likely antimicrobial ability of SpCrus2 may result from its microbial polysaccharide-binding and agglutination activities. Overall, this study characterized the first type II crustin in crabs and provided new insights into understanding the sequence and functional diversity of crustins and their immune functions in crustaceans.

Newly identified invertebrate-type lysozyme (Splys-i) in mud crab (Scylla paramamosain) exhibiting muramidase-deficient antimicrobial activity.

Lysozymes are widely distributed immune effectors exerting muramidase activity against the peptidoglycan of the bacterial cell wall to trigger cell lysis. However, some invertebrate-type (i-type) lysozymes deficient of muramidase activity still exhibit antimicrobial activity. To date, the mechanism underlying the antimicrobial effect of muramidase-deficient i-type lysozymes remains unclear. Accordingly, this study characterized a novel i-type lysozyme, Splys-i, in the mud crab Scylla paramamosain. Splys-i shared the highest identity with the Litopenaeus vannamei i-type lysozyme (Lvlys-i2, 54% identity) at the amino acid level. Alignment analysis and 3D structure comparison show that Splys-i may be a muramidase-deficient i-type lysozyme because it lacks the two conserved catalytic residues (Glu and Asp) that are necessary for muramidase activity. Splys-i is mainly distributed in the intestine, stomach, gills, hepatopancreas, and hemocytes, and it is upregulated by Vibrio harveyi or Staphylococcus aureus challenge. Recombinant Splys-i protein (rSplys-i) can inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio alginolyticus, Vibrio parahemolyticus, and Escherichia coli), Gram-positive bacteria (S. aureus, Bacillus subtilis, and Bacillus megaterium), and the fungus Candida albicans to varying degrees. In this study, two binding assays and a bacterial agglutination assay were conducted to elucidate the potential antimicrobial mechanisms of Splys-i. Results demonstrated that rSplys-i could bind to all nine aforementioned microorganisms. It also exhibited a strong binding activity to lipopolysaccharide from E. coli and lipoteichoic acid and peptidoglycan (PGN) from S. aureus but a weak binding activity to PGN from B. subtilis and ß-glucan from fungi. Moreover, rSplys-i could agglutinate these nine types of microorganisms in the presence of Ca2+ at different protein concentrations. These results suggest that the binding activity and its triggered agglutinating activity might be two major mechanisms of action to realize the muramidase-deficient antibacterial activity. In addition, rSplys-i can hydrolyze the peptidoglycan of some Gram-positive bacteria because it exhibits weak isopeptidase activities in salt and protein concentration-dependent manner. This result indicates that such an isopeptidase activity may contribute to the muramidase-deficient antimicrobial activity to a certain degree. In conclusion, Splys-i is upregulated by pathogenic bacteria, and it inhibits bacterial growth by binding and agglutination activities as well as isopeptidase activity, suggesting that Splys-i is involved in immune defense against bacteria through several different mechanisms of action.

SpALF4: a newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity.

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand ß-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.

Identification, characterization, and functional analysis of Tube and Pelle homologs in the mud crab Scylla paramamosain.

Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria.

A novel peroxinectin involved in antiviral and antibacterial immunity of mud crab, Scylla paramamosain.

Peroxinectin (PX) with cell adhesion and peroxidase activities is important in invertebrate immune responses. We identified a novel PX homolog from Scylla paramamosain (designated as Sp-PX) through transcriptome sequencing. The full-length of cDNA sequence was 3,165 bp. And there was a peroxidase domain in the deduced protein sequence. A cell-adhesive sequence (KGD motif) was also found in the N-terminus. The predicted molecular mass of the mature protein is 83.9 kDa, with an estimated pI of 6.21. At the amino acid level, Sp-PX shared much higher similarities with other crustaceans PX proteins. And Sp-PX also exhibited some similarities with other peroxidase family members. According to real-time polymerase chain reaction, Sp-PX was mainly distributed in the hemocytes. The gene expression levels in the hemocytes of the normal and white spot syndrome virus (WSSV)-challenged crabs were compared via high-throughput RNA sequencing technology, and the results showed that Sp-PX was upregulated at 48 h post-WSSV challenge. Subsequently, how Sp-PX responds to WSSV stimulus was explored through time-course experiments. The Sp-PX transcripts dramatically increased and reached the highest level at 12 h post-injection, whereas Sp-PX transcripts were recovered at 96 h post-challenge. Meanwhile, it was found that the WSSV copies proliferated significantly after a period of latent viral infection for 48 h. In addition,Sp-PX transcripts were also upregulated after Vibrio harveyi or Staphylococcus aureus challenge. Overall, Sp-PX not only participates in antibacterial immunity but also plays a crucial role in the antiviral immune responses of mud crab at the early stage of WSSV infection.

Inhibition of CYP450 1A and 3A by berberine in crucian carp Carassius auratus gibelio.

Berberine has long been considered as an antibiotic candidate in aquaculture. However, studies regarding its effects on drug-metabolizing enzymes in fish are still limited. In the present study, the effects of berberine on cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injection of different concentrations of berberine (0, 5, 25, 50, and 100mg/kg) inhibited the CYP1A mRNA expression, thereby inhibiting further the catalytic activity of CYP1A-related ethoxyresorufin-O-deethylase (EROD). Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing berberine concentrations. In addition, the CYP3A expressions at both the mRNA and the protein levels were downregulated by higher berberine concentrations. The catalytic activity of CYP3A-related erythromycin N-demethylase (ERND) was also inhibited by berberine at a dose of no less than 25mg/kg. Moreover, at the berberine concentration exceeding 25mg/kg, the inhibition of CYP3A expression and ERND activity increased with increasing berberine concentrations. In vitro experiments were also performed. When berberine was pre-incubated with the crucian carp liver microsomes, it competitively inhibited the corresponding EROD activity with the IC(50) of 11.7 µM. However, the ERND activity was slightly inhibited by berberine with the IC(50) of 206.4 µM. These results suggest that, in crucian carp, berberine may be a potent inhibitor to CYP1A, whereas the CYP3A inhibition needs a higher concentration of berberine.

Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene.

Transmissible gastroenteritis virus (TGEV) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas. Up to date, various vaccines developed against TGEV still need to be improved. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, two shRNA-expressing plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) targeting the RNA-dependent RNA polymerase (RdRp) gene of TGEV were constructed and transfected into swine testicular (ST) cells. The cytopathic effect (CPE) and MTS assays demonstrated that both shRNAs were capable of protecting cells against TGEV invasion with very high specificity and efficiency. A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the two plasmids were reduced by 95.2% and up to 100%, respectively. Our results suggest that RNAi might be a promising new strategy against TGEV infection.