A complement factor I (CFI) gene mediates innate immune responses in yellow catfish Pelteobagrus fulvidraco.

This study isolated CFI gene from Pelteobagrus fulvidraco and named it PfCFI. The cDNA of PfCFI is 2374 bp long, including a 52 bp 5' untranslated sequence, a 222 bp 3' untranslated sequence, and an open reading frame (ORF) of 2100 bp encoding polypeptide consisting of 699 amino acids. Phylogenetic analysis revealed that the PfCFI was closely related to CFI of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis indicate that there is the PfCFI gene which expressed in all the rest of tested tissues in varied levels, and mainly distributed in liver and least in heart. The reseachers induce the expressions level of PfCFI gene in liver, spleen, head kidney and blood at different points in time after challenged with lipopolysaccharide (LPS), and polyriboinosinic polyribocytidylic acid (poly I:C), respectively. Together these results suggested that CFI gene plays an important role in resistance to pathogens in yellow catfish immunity.

1 H NMR-based metabonomic evaluation of the pesticides camptothecin and matrine against larvae of Spodoptera litura.

BACKGROUND: Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. RESULTS: CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. CONCLUSIONS: These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.

The red swamp crayfish, Procambarus clarkii cathepsin C, participates in the innate immune response to the viral and bacterial pathogens.

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys248, His395 and Asn417). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans.

A non-mammalian Toll-like receptor 26 (TLR26)gene mediates innate immune responses in yellow catfish Pelteobagrus fulvidraco.

In this study, we identified a fish-specific Toll-like receptor (TLR) in Pelteobagrus fulvidraco, an economically important freshwater fish in China. This TLR, PfTLR26, was shown to be encoded by a 3084 bp open reading frame (ORF), producing a polypeptide 1027 amino acids in length. The PfTLR26 protein contains a signal peptide, eight leucine-rich repeat (LRR) domains, two LRR_TYP domains in the extracellular region, and a Toll/interleukin (IL)-1 receptor (TIR) domain in the cytoplasmic region, consistent with the characteristic TLR domain architecture. This predicted 117.1 kDa protein was highly homologous to those of other fish, with phylogenetic analysis revealing the closest relation to TLR26 of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PfTLR26 gene was expressed in all tissues tested, with the highest expression levels seen in the head kidney and blood, and the lowest seen in muscle. PfTLR26 exhibited significant upregulation in liver, spleen, head kidney, and blood at different time points following challenge with the common TLR agonists lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). Taken together, these results suggest that PfTLR26 may be an important component of the P. fulvidraco innate immune system, participating in the transduction of TLR signaling under pathogen stimulation.

Identification and molecular characterization of a novel anti-lipopolysaccharide factor (ALF) from red swamp crayfish, Procambarus clarkii.

Anti-lipopolysaccharide factors are a group of small proteins with broad spectrum antiviral property and antibacterial activity. Herein, we obtained the genomic sequence of the Procambarus clarkii anti-lipopolysaccharide factor (PcALF) gene by using polymerase chain reaction to investigate its expression pattern in various tissues and in the immune tissues (Hepatopancreas) following exposure to pathogens. The deduced protein of PcALF was conserved; it displayed the signal peptides and putative lipo-polysaccharide binding domain, particularly the two conserved cysteine amino acid residues at both ends of the domain. The recombinant protein of PcALF was successfully expressed in Escherichia coli and rabbit anti-PcALF polyclonal antibodies were prepared. The qRT-PCR analysis showed unequal distribution of PcALF transcript in the examined tissues, however the transcript level was greatest in hepatopancreas. The challenge with peptidoglycan (PGN), lipo-polysaccharide (LPS) and Poly I:C significantly enhanced expression level of PcALF in hepatopancreas when compared with the PBS control. RNA interference of PcALF affected the mRNA expression levels of immune-related genes. Taken together, our data suggested that PcALF is an inducible protein and could play a key biological role in the innate immune defense of P. clarkii.

Transcriptome Analysis Reveals Potential Antioxidant Defense Mechanisms in Antheraea pernyi in Response to Zinc Stress.

The growth and development of the Chinese oak silkworm, Antheraea pernyi, are strongly influenced by environmental conditions, including heavy metal pollution. An excess of heavy metals causes cellular damage through the production of free radical reactive oxygen species. In this study, transcriptome analysis was performed to investigate global gene expression when A. pernyi was exposed to zinc infection. With RNA sequencing (RNA-Seq), a total of 25 795 510 and 38 158 855 clean reads were obtained from zinc-treated and control fat body libraries, respectively. We identified 2399 differential expression genes (DEGs) (1845 upregulated and 544 downregulated genes) in the zinc-treated library. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were related to the peroxisome pathway that was associated with antioxidant defense. Our results suggest that fat bodies of A. pernyi constitute a strong antioxidant defense against heavy metal contamination.

Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipopolysaccharide (LPS) infection.

The RNA-sequencing followed by de novo assembly generated 61,912 unigene sequences of P. clarkii hepatopancreas. Comparison of gene expression between LPS challenged and PBS control samples revealed 2552 differentially expressed genes (DEGs). Of these sequences, 1162 DEGs were differentially up-regulated and 1360 DEGs differentially down-regulated. The DEGs were then annotated against gene ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Some immune-related pathways such as PPAR signaling pathway, lysosome, Chemical carcinogenesis, Peroxisome were predicted by canonical pathways analysis. The reliability of transcriptome data was validated by quantitative real time polymerase chain reaction (qRT-PCR) for the selected genes. The data presented here shed light into antibacterial immune responses of crayfish. In addition, these results suggest that transcriptomic data provides valuable sequence resource for immune-related gene identification and helps to understand P. clarkii immune functions.

Characterization and function of a cathepsin B in red crayfish (Procambarus clarkii) following lipopolysaccharide challenge.

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In the present study, a cathepsin B gene (named PcCTSB) was cloned and characterized from the red crayfish, Procambarus clarkii. The cDNA fragments of PcCTSB was 990 bp in length. It encoded a putative protein of 329 amino acid residues with predicted molecular weight of 36.4 kDa and isoelectric point of 7.020. Sequence alignment revealed that PcCTSB protein is 53.6%-80.4% identical with those from other 10 species. The predicted tertiary structure of PcCTSB protein was highly similar to that of animals. The results of the phylogenetic analysis indicated that the PcCTSB protein could be clustered with the Eriocheir sinensis cathepsin B protein. The recombinant protein of PcCTSB was expressed successfully in Escherichia coli cells. The mRNA expressions of PcCTSB were detected in all tested tissues, particularly high in the hepatopancreas. After lipopolysaccharide (LPS) challenge, the expression levels of PcCTSB were up-regulated significantly at different time points compared with control. Our results suggested that the PcCTSB might play an important role in defending against the pathogenes infection.

Molecular characterization of an Apolipophorin-III gene from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

Apolipophorin-III (ApoLp-III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp-III gene of Antheraea pernyi pupae (Ap-ApoLp-III) was isolated and characterized. The full-length cDNA of Ap-ApoLp-III is 687 bp, including a 5'-untranslated region (UTR) of 40 bp, 3'-UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin-III precursor domain (PF07464). The deduced Ap-apoLp-III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap-apoLp-III was close to that of Bombycoidea. qPCR analysis revealed that Ap-ApoLp-III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap-ApoLp-III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap-ApoLp-III showed that the expression of Ap-ApoLp-III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap-ApoLp-III gene acts in the innate immunity of A. pernyi.

Characterization of the complete mitochondrial genome of Diaphania pyloalis (Lepidoptera: Pyralididae).

The complete mitochondrial genome (mitogenome) of Diaphania pyloalis (Lepidoptera: Pyralididae) was determined to be 15,298 bp and has the typical gene organization of mitogenomes from lepidopteran insects. It consists of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A+T-rich region. The A+T content of this mitogenome is 80.83% and the AT skew is slightly positive. All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which is initiated by CGA. Only the cox2 gene has an incomplete stop codon consisting of just a T. All the tRNA genes display a typical clover-leaf structure of mitochondrial tRNA. The A+T-rich region of the mitogenome is 332 bp in length, including several common features found in lepidopteran mitogenomes. Phylogenetic analysis showed that the D. pyloalis is close to Pyralididae.

Overexpression of small heat shock protein 21 protects the Chinese oak silkworm Antheraea pernyi against thermal stress.

Small heat shock proteins (sHSPs) usually act as molecular chaperones to prevent proteins from being denatured in extreme conditions. We first report the sHSP21 gene, named as Ap-sHSP21, in the Chinese oak silkworm Antheraea pernyi (Lepidoptera: Saturniidae). The full-length cDNA of Ap-sHSP21 is 976 bp, including a 5'-untranslated region (UTR) of 99 bp, a 3'-UTR of 316 bp and an open reading frame (ORF) of 561 bp encoding a polypeptide of 186 amino acids. The deduced A. pernyi sHSP21 protein sequence reveals the percent identity is 82-93% in comparison to other sHSPs from insects. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis shows that Ap-sHSP21 expression is higher in testis than that in other examined tissues and significantly up-regulated after heat shock. In addition, prokaryotic expression and purification of the Ap-sHSP21 protein were performed. SDS-PAGE and Western blot analysis demonstrated that a 25 kDa recombinant protein was successfully expressed in Escherichia coli cells and the purified recombinant protein was also confirmed to protect restriction enzymes from thermal inactivation. The expression of Ap-sHSP21 was significantly down-regulated after RNA interference, which was confirmed by qRT-PCR and Western blot analysis. All together, these results suggest that Ap-sHSP21 play a key role in thermal tolerance.