Xin-Cang Li

A nonluminescent and highly virulent Vibrio harveyi strain is associated with "bacterial white tail disease" of Litopenaeus vannamei shrimp.

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by “white tail” and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of “white tail” but of non-bacterial origin, the present disease was named as “bacterial white tail disease (BWTD)”. Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.

A novel myeloid differentiation factor 88 homolog, SpMyD88, exhibiting SpToll-binding activity in the mud crab Scylla paramamosain

Myeloid differentiation factor 88 (MyD88) is an essential regulator in the Toll or Toll-like receptor (TLR) signaling pathway. In the current study, we characterized a novel crustacean MyD88 homolog, SpMyD88, and analyzed its binding activity with SpToll. The full-length cDNA sequence of SpMyD88 is 2933 bp, with a 1419 bp open reading frame encoding a 472-amino acid protein. No signal peptide was predicted. A death domain (residues 19-103), a Toll/interleukin-1 receptor (TIR) domain (residues 156-297), and a C-terminal extension (CTE) domain (residues 298-472) were also found. In a phylogenetic tree constructed with MyD88 homologs from both invertebrates and vertebrates, arthropod MyD88s including SpMyD88 formed a cluster containing a unique CTE domain. SpToll shared the highest identity with human TLR4. These two receptors were grouped into a cluster of a tree constructed based on the conserved TIR domains. SpToll also had a close relationship with other shrimp TLRs that possess potential antibacterial activity. SpMyD88 was highly expressed in the hemocytes, gills, hepatopancreas, and eye stalks. Upon challenge with Vibrio harveyi, both SpMyD88 and SpToll were significantly increased in the hemocytes, whereas only SpMyD88 was elevated by Staphylococcus aureus. In addition, a pull-down assay demonstrated that SpMyD88 showed a binding activity with SpToll. These results suggest that SpMyD88 and SpToll are involved in the defense system of mud crabs against Gram-negative bacteria.

A novel anti-lipopolysaccharide factor SpALF6 in mud crab Scylla paramamosain exhibiting different antimicrobial activity from its single amino acid mutant

Abstract In crustaceans, anti‐lipopolysaccharide factors (ALFs) are important immune effectors that have sequence diversity and exhibit broad antimicrobial activities. In this study, we characterized a novel ALF homolog SpALF6 from mud crab Scylla paramamosain and its variant SpALF6‐V, which was generated by mutations of two amino acids (H46 to R and A110 to P) due to the presence of two single nucleotide polymorphisms (SNPs). SpALF6 was an anionic peptide with isoelectric point (pI) 6.79, whereas SpALF6‐V was a cationic protein with pI 7.98. These two proteins shared a common lipopolysaccharide (LPS)‐binding domain (LBD) with pI 6.05. SpALF6 was expressed mainly in hemocytes and up‐regulated by Vibrio parahaemolyticus or Staphylococcus aureus challenge, indicating that SpALF6 may participate in the antibacterial immune responses. To investigate the likely functional differences between SpALF6 and SpALF6‐V and elucidate the underlying mechanisms, a single amino acid mutant SpALF6‐M (from H46 to R, outside but very close to LBD), which had the same pI as SpALF6‐V, was harvested by a fusion PCR. Then, both SpALF6 and SpALF6‐M were overexpressed and purified to test antimicrobial activity and binding activity to microbial cells or polysaccharides. SpALF6‐M exhibited more potent antimicrobial and cell‐binding activity on Gram‐positive bacteria and fungi than SpALF6. Furthermore, SpALF6‐M possessed stronger lipoteichoic acid (LTA)‐binding activity than SpALF6, demonstrating that this particular positively charged amino acid outside but close to LBD contributed to the increase in SpALF6‐M antibacterial activity. In addition, SpALF6 LBD peptide and its biotin‐labeled form were synthesized in this study. Results showed that this anionic LBD peptide itself did not exhibit any significant antimicrobial activity against 10 kinds of microorganisms but it possessed strong binding activity to LPS, LTA, and peptidoglycan. These findings suggested that this anionic LBD was still an important active center and required collaboration with some particular positively charged amino acids outside LBD to exhibit antibacterial activity. Thus, SpALF6‐M antimicrobial activity was increased by the mutation of H46 to R instead of A110 to P, which did not change the protein charge, suggesting that SpALF6‐V may have more potent antimicrobial activity than SpALF6 and play more important roles in antibacterial immunity. This study provided a new insight into the mechanisms of how ALF amino acid sequence diversity resulted in their functional divergence. HighlightsWe characterized a novel ALF homolog SpALF6 and its variant SpALF6‐V.SpALF6‐M having the same pI as SpALF6‐V was produced by mutation of H46 in SpALF6 to R.SpALF6‐M possessed stronger antimicrobial and LTA‐binding activities than SpALF6.The anionic LBD of SpALF6 did not exhibit any antimicrobial activity.The SNP locus of SpALF6‐V can be a selective marker of disease resistance in breeding.

Pharmacokinetics and Tissue Distribution of Thiamphenicol and Florfenicol in Pacific White Shrimp Litopenaeus vannamei in Freshwater following Oral Administration

This study evaluated the pharmacokinetic disposition of thiamphenicol (THA) and florfenicol (FLR) after oral administration of each at a single dose of 10 mg/kg body weight in Pacific white shrimp Litopenaeus vannamei held in freshwater at 25.0 ± 1.0°C. The THA and FLR concentrations in the hemolymph, muscle, and hepatopancreas were determined by HPLC. The profiles of hemolymph THA and FLR concentrations versus time were best described by a two-compartment open pharmacokinetic model with first-order absorption. The peak concentration (C max), peak time (T max), absorption half-life (t 1/2ka) and elimination half-life (t 1/2β) of THA in hemolymph were 7.96 μg/mL, 2 h, 0.666 h, and 10.659 h, respectively. The corresponding values for FLR were 5.53 μg/mL, 2 h, 1.069 h, and 17.360 h, respectively. After oral administration, THA and FLR were rapidly absorbed in white shrimp and THA in hemolymph was absorbed and eliminated more quickly than FLR. The parameters in muscle and hepatopancreas were calculated by a noncompartment model based on statistical moment theory. The C max, area under the concentration-time curve (AUC0-t ), mean residue time (MRT0-t ), and half-life (t 1/2z) in muscle were 2.98 μg/g, 29.10 mg/kg·h, 9.77 h, and 6.84 h, respectively. The corresponding values for FLR were 1.91 μg/g, 15.97 mg/kg·h, 19.40 h, and 18.32 h, respectively. In muscle THA was eliminated more quickly than FLR. The peak concentrations of THA and FLR in the hepatopancreas were 204.25 and 164.22 μg/g, respectively, and the values for AUC0-t were 1,337.74 and 871.73 mg/kg·h, respectively, which were much higher than those in hemolymph and muscle. The in vitro protein-binding value of THA (28.38%) was lower than that of FLR (37.91%), which might be related to the finding that THA in Pacific white shrimp was absorbed and eliminated more quickly than FLR. Received July 21, 2012; accepted November 23, 2012.

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.

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.

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

&NA; 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 &bgr;‐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. HighlightsSplys‐i is a muramidase‐deficient i‐type lysozyme with antimicrobial activity.Splys‐i possesses binding activity to microbes and their specific polysaccharides.Splys‐i can agglutinate nine different types of bacteria and fungi.Splys‐i exhibits isopeptidase activity in salt and protein concentration‐dependent way.Agglutination and isopeptidase activity contribute to Splys‐i antimicrobial activity.

PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the nucleus

Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine transcription factor PcDorsal, into the nucleus participating in the immune defense against Gram-negative bacteria.

Newly identified type II crustin ( Sp Crus2) in Scylla paramamosain contains a distinct cysteine distribution pattern exhibiting broad antimicrobial activity

&NA; 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.

Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii

C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense.