Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factor 6 (traf6) like gene involved in antibacterial innate immune of fresh water crayfish, Procambarus clarkii.

In invertebrates, innate immunity was the crucial defending pattern against pathogenic microorganisms. For the past few years, Toll or Toll like receptors (TLRs) signaling pathway was studied extensively in crustaceans. Among the components of Toll or Toll like receptors (TLRs) signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6) acted as an important cytoplasmic adaptor, which was conserved from Drosophila to human. In this study, a new traf6 like gene was cloned from hepatopancreas of P. clarkii. After challenged respectively by S. aureus or E. ictaluri, the expression profiles were studied. And the results showed that the mRNA transcript of Pc-traf6 like gene was up-regulated significantly in the hemocytes, hepatopancreas, gills, and intestine of crayfish. After Pc-traf6 like gene was knocked down, the expression levels of transcription factor (Dorsal) and some crucial immunity effectors (ALF 3, Lysozyme 1, Lectin 1, and Crustin 2) in TLRs signaling pathway were dramatically suppressed. Simultaneously, the survival rate of crayfish challenged respectively by S. aureus or E. ictaluri was significantly decreased in RNAi assay. All these results indicated that Pc-traf6 like gene played an important role in regulating the expression of downstream effectors in the TLRs signaling pathway of crayfish.

A new antimicrobial peptide isoform, Pc-crustin 4 involved in antibacterial innate immune response in fresh water crayfish, Procambarus clarkii.

The main advantage of antimicrobial peptides (AMPs) used as the effectors in the innate immunity system of invertebrates is that the high specificity is not indispensable. And they play important roles in the systemic defenses against microbial invasion. In this study, a new full-length cDNA of the crustins molecule was identified in red swamp crayfish, P. clarkii (named Pc-crustin 4). The ORF of Pc-crustin 4 contained 369 bp which encoded a protein of 122 amino acids, with a 20-amino-acid signal peptide sequence. On the base of the classification method established by Smith et al., Pc-crustin 4 belonged to Type Ⅰ crustin molecule. The Pc-crustin 4 transcripts were expressed in hemocytes at relatively high level, and relatively low level in hepatopancreas, gills, and intestine in normal crayfish. After respectively challenged with S. aureus or E. ictaluri, the expression levels of Pc-crustin 4 showed up-regulation trends at different degrees in the hemocytes, hepatopancreas, gills, and intestine tissues. Besides, the results of liquid antibacterial assay showed that rPc-crustin 4 inhibited obviously the growth of S. aureus and E. ictaluri. The results of bacteria binding assay showed that rPc-crustin 4 could bind strongly to S. aureus and E. ictaluri. Finally, RNAi assay was performed to study the immunity roles of Pc-crustin 4 in crayfish in vivo. Taken together, Pc-crustin 4 is an important immunity effector molecule, which plays crucial roles in defending against bacterial infection in crayfish.

Molecular cloning, expression and biological activity of rhesus macaque interleukin-17A and interleukin-17F.

Interleukin-17A (IL-17A) and interleukin-17F (IL-17F) as two potent proinflammatory cytokines and the signature cytokines of Th17 cells play important roles in human autoimmune diseases, inflammation and host defenses. In this study, rhesus macaque IL-17A (rhIL-17A) and IL-17F (rhIL-17F) were cloned and expressed, and their biological activities and in vivo distribution were examined. The resulting data showed that both the rhIL-17A and rhIL-17F genes were consisted of three exons and two introns. RhIL-17A and rhIL-17F shared 96.8% and 93.9% amino acid sequence identity with human IL-17A (huIL-17A) and IL-17F (huIL-17F) respectively and the sequences also shared one N-glycosylation site and six conserved cysteine residues with huIL-17A and huIL-17F. IL-17A and IL-17F transcripts were highly expressed in lymphoid tissues and the intestinal tract of rhesus macaques. Functionally, recombinant rhIL-17A and rhIL-17F showed similar effect on Act1 levels and NF-κB phosphorylation compared with that of commercial human IL-17A and IL-17F. Moreover, the antibacterial proteins (such as ß-defensin 2, S100A8, S100A9, RegIIIα and Muc1) and the tight junction associated genes (including CLDN1, CLDN4, OCLN, and ZO1) expressed by Caco-2 cells were largely enhanced after treatment with rhIL-17A and rhIL-17F. Meanwhile, purified rhIL-17A and rhIL-17F could also induce the expression of IL-6 and TNF-α by THP-1 cells. These data indicated that rhesus macaque IL-17A and IL-17F are highly similar to that of humans in both structure and function. Studies on rhIL-17A/rhIL-17F are promising approach to contribute to the understanding of human IL-17A and IL-17F-related intestinal diseases.

Molecular characterization and expression analysis of dopa decarboxylase involved in the antibacterial innate immunity of the freshwater crayfish, Procambarus clarkii.

Dopa decarboxylase (DDC) is responsible for the synthesis of dopamine, which acts as an important modulator in the nervous systems of vertebrates and invertebrates. Recent studies have indicated that DDC also plays crucial roles in the insect innate immune system. However, the functions of DDC in immunomodulation in crustaceans have not been thoroughly elucidated to date. In this study, a new full-length cDNA of the DDC protein was identified from red swamp crayfish, Procambarus clarkii (named Pc-ddc). The ORF of Pc-ddc encoded 474 amino acids, which possessed a 377-amino-acid domain. Pc-ddc was expressed at a relatively high level in the hemocytes and gills of crayfish. This protein was expressed at a relatively low level in the hepatopancreas and intestine. The expression level of Pc-ddc was clearly upregulated in hemocytes, hepatopancreas, gills, and intestine tissues after challenge with S. aureus or E. ictaluri. The results of the enzyme catalysis assay showed that the enzyme catalysis activity of rPc-DDC was 35 ±â€¯2.8 ng h-1 mg-1 (n = 3). In addition, the results of the mimetic crayfish hemocytes encapsulation assay showed that the encapsulation rate of beads coated with rPc-DDC was clearly increased. The results of the bacterial binding assay showed that rPc-DDC strongly binds to S. aureus and E. ictaluri. Finally, when Pc-ddc was knocked down, the number of surviving crayfish clearly decreased after S. aureus or E. ictaluri was injected. All of these results indicate that Pc-DDC is an important immunomodulating enzyme in the neuroendocrine-immune (NEI) system of crayfish.

A new crustin homologue (SpCrus6) involved in the antimicrobial and antiviral innate immunity in mud crab, Scylla paramamosain.

Crustins play important roles in defending against bacteria in the innate immunity system of crustaceans. In present study, we identified a crustin gene in Scylla paramamosain, which was named as SpCrus6. The ORF of SpCrus6 possessed a signal peptide sequence (SPS) at the N-terminus and a WAP domain at the C-terminus. And there were 5 Proline residues, 5 Glycine and 4 Cysteine residues between SPS and WAP domain in SpCrus6. These features indicated that SpCrus6 was a new member of crustin family. The SpCrus6 mRNA transcripts were up-regulated obviously after bacteria or virus challenge. These changes showed that SpCrus6 was involved in the antimicrobial and antiviral responses of Scylla paramamosain. Recombinant SpCrus6 (rSpCrus6) showed strong inhibitory abilities against Gram-positive bacteria (Bacillus megaterium, Staphylococcus aureus, and Bacillus subtilis). But the inhibitory abilities against four Gram-negative bacteria (Vibrio parahemolyticus, Vibrio alginolyticus, Vibrio harveyi and Escherichia coli) and two fungi (Pichia pastoris and Candida albicans) were not strong enough. Besides, rSpCrus6 could strongly bind to two Gram-positive bacteria (B. subtilis and B. megaterium) and three Gram-negative bacteria (V. alginolyticus, V. parahemolyticus, and V. harveyi). And the binding levels to S. aureus and two fungi (P. pastoris and C. albicans) were weak. The polysaccharides binding assays' results showed rSpCrus6 had superior binding activities to LPS, LTA, PGN and ß-glucan. Through agglutinating assays, we found rSpCrus6 could agglutinate well three Gram-positive bacteria (S. aureus, B. subtilis and B. megaterium). And the agglutinating activities to Gram-negative bacteria and fungi were not found. In the aspect of antiviral functions, rSpCrus6 could bind specifically to the recombinant envelop protein 26 (rVP26) of white spot syndrome virus (WSSV) but not to recombinant envelop protein 28 (rVP28), whereas GST protein could not bind to rVP26 or rVP28. Besides, rSpCrus6 could suppress WSSV reproduction to some extent. Taken together, SpCrus6 was a multifunctional immunity effector in the innate immunity defending response of S. paramamosain.

High-throughput sequencing analysis of microRNAs in gills of red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

MicroRNAs (miRNAs) are important posttranscriptional regulators. They play an important role in the antiviral innate immunity of invertebrates. In the present study, high-throughput small RNAs Illumina sequencing systems were carried out to identify differentially expressed miRNAs (DEMs) in the gills of Procambarus clarkii, which was challenged with white spot syndrome virus (WSSV). Our results identified 11,617 known and 6 novel miRNAs in normal group (NG) and WSSV-challenged group (WG) small RNA libraries. Additionally, 27 DEMs were shown to participate in the antiviral innate immunity of P. clarkii and were significantly upregulated or downregulated. In addition, the results of the KEGG pathway prediction of the DEMs target genes showed that putative target genes of these 27 DEMs were related mainly to the RNA transport pathway, tight junction pathway, mRNA surveillance pathway, regulation actin cytoskeleton pathway, focal adhesion pathway, and MAPK signaling pathway. These results provide important information for future studies about the antiviral innate immunity of crustaceans.

Identification and characterization of intestine microRNAs and targets in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.

Identification and characterization of lymph organ microRNAs in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

MicroRNAs (miRNAs) were important post-transcriptional regulators and played vital roles in innate immunity system of invertebrates, especially in the aspect of antivirus. In this study, using high-throughput small RNAs Illumina sequencing system, differentially expressed miRNAs (DEMs) from lymph organs in red swamp crayfish, Procambarus clarkii, infected with white spot syndrome virus, were identified. As a result, 32 known miRNAs and 7 novel miRNAs were identified in crayfish lymph organ small RNAs library of NG and WG. Among them, 7 differentially expressed miRNAs (DEMs) were predicted to be involved in the lymph organ antiviral innate immunity of P. clarkii. Besides, the results showed that putative target genes of these DEMs were related with tight junction, RNA transport, regulation of actin cytoskeleton, focal adhesion, vascular smooth muscle contraction, mRNA surveillance pathway, NOD-like receptor signaling pathway, leukocyte transendothelial migration, and protein processing in endoplasmic reticulum. These results might provide the guiding theoretical foundation for future studies about crustaceans' antiviral innate immunity.

[Simultaneous determination of 6 neonicotinoid residues in soil using DLLME-HPLC and UV].

A simple, cheap and rugged method was developed for simultaneous deter mination of 6 neonicotinoid residues in soil, including imidacloprid, acetamiprid, thiamethoxam, thiacloprid, clothianidin and nitenpyram. The soil sample was produced by dispersive liquid-liquid micro-extraction (DLLME) after extracted by the mixed solution of acetonitrile and CH2Cl2 (2:1, phi). The analytes were separated by HPLC with Alltima C18 column (4.6 mm x 250 mm, 5 microm) and detected by PDA at 260 nm. External standard method was used for quantification. The results showed that good linearity was obtained with correlation coefficients between 0.9982 and 0.9999 in the range of 0.5-200 microg x L(-1). The limits of detection (LODs) were in the range between 0.0005 and 0.003 microg x mL(-1) (S/N = 3). The method was validated with five soil samples spiked at three fortification levels (0.05, 0.1, 1.0 mg x kg(-1)) and recoveries were in the range of 55.3%-95.6% with RSD of 1.4%-7.0%. The effect of clean-up was evaluated by UV spectra and demonstrated that the method established is effective. In conclusion, this method is competent for the simultaneous analysis of 6 neonicotinoid residues in soil.