Ferritin Heavy-like subunit is involved in the innate immune defense of the red swamp crayfish Procambarus clarkii.

Iron-binding proteins, known as ferritins, play pivotal roles in immunological response, detoxification, and iron storage. Despite their significance to organisms, little is known about how they affect the immunological system of the red swamp crayfish (Procambarus clarkii). In our previous research, one ferritin subunit was completely discovered as an H-like subunit (PcFeH) from P. clarkii. The full-length cDNA of PcFerH is 1779 bp, including a 5'-UTR (untranslated region, UTR) of 89 bp, 3'-UTR (untranslated region, UTR) of 1180 bp and an ORF (open reading frame, ORF) of 510 bp encoding a polypeptide of 169 amino acids that contains a signal peptide and a Ferritin domain. The deduced PcFerH protein sequence has highly identity with other crayfish. PcFerH protein's estimated tertiary structure is quite comparable to animal structure. The PcFerH is close to Cherax quadricarinatus, according to phylogenetic analysis. All the organs examined showed widespread expression of PcFerH mRNA, with the ovary exhibiting the highest levels of expression. Additionally, in crayfish muscles, intestines, and gills, the mRNA transcript of PcFerH was noticeably up-regulated, after LPS and Poly I:C challenge. The expression of downstream genes in the immunological signaling system was suppressed when the PcFerH gene was knocked down. All of these findings suggested that PcFerH played a vital role in regulating the expression of downstream effectors in the immunological signaling pathway of crayfish.

The analyses of the complete mitochondrial genomes of three crabs revealed novel gene rearrangements and phylogenetic relationships of Brachyura.

BACKGROUND: Brachyura crab is the largest branch of Decapoda crustacean. Phylogenetic relationships within Brachyura remain controversial to be investigated. The mitochondrial genome (mitogenome) is an important molecular marker for studying the phylogenetic relationships of Brachyura. METHODS AND RESULTS: To understand the phylogeny of Brachyura, the three complete mitogenomes from Charybdis annulata, Leptodius exaratus, and Spider crab were sequenced and annotated. Their full length was 15,747, 15,716, and 16,608 bp long, respectively. The first two crabs both contained 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. However, Spider crab contained 13 PCGs, two rRNA genes, 25 tRNA genes and a control region. The mitogenomes of each of the three crabs exhibited high AT content (67.8%, 69.1%, and 70.8%), with negative AT skews (-0.014, - 0.028, and - 0.017) and GC skews (-0.269, - 0.286, and - 0.341). The gene order of C. annulata was identical to the ancestor of Brachyura. Compared with the ancestor of Brachyura, L. exaratus exhibited the gene rearrangements of Val (V)-rrnS-control region, and Spider crab had the four copies of Lys (K). Phylogenetic analyses indicated that C. annulata belonged to Portunidae family, Portunoidea superfamilies, L. exaratus belonged to Xanthidae family, Xanthoidea superfamilies, and Spider crab belonged to Mithracidae family, Majoidea superfamilies. Phylogenetic analyses showed that the two species (Somanniathelphusa boyangensis and Huananpotamon lichuanense) belonging to the Potamoidea were sister groups to the Thoracotremata, thus supporting the conclusion that Heterotremata is polyphyletic. CONCLUSION: The results of this study enriched the crab mitogenome database and enabled us to better understand the phylogenetic relationships of Brachyura.

Transcriptome analysis reveals antioxidant defense mechanisms in the red swamp crayfish Procambarus clarkia after exposure to chromium.

Chromium (Cr) as a chromate anion has a strong redox capacity that seriously threatens the ecological environment and human health. Cr can contaminate water and impart toxicity to aquatic species. Procambarus clarkii is an important food source that once represented a large proportion of the aquaculture industry due to its rapid reproduction and high economic value. However, there have been reports on the death of P. clarkii due to heavy metal pollution. The underlying mechanism regarding heavy metal toxicity was studied in this paper. The transcriptome data of hemocytes extracted from P. clarkii injected with Cr were analyzed by high-throughput sequencing and compared to the control group. In total, 48,128,748 clean reads were obtained in the treatment group and 56,480,556 clean reads were obtained in the control group. The reads were assembled using Trinity and the identified unigenes were then annotated. Then, 421 differentially-expressed genes (DEGs) were found, 170 of which were upregulated and 251 downregulated. Many of these genes were found to be related to glutathione metabolism and transportation. The glutathione metabolic pathway of P. clarkii was thus activated by Cr exposure to detoxify and maintain body function. Validation of DEGs with quantitative real-time PCR confirms the changes in gene expression. Thus, this study provides data supporting a glutathione-focused response of P. clarkii to exposure to heavy metals.

Differentially expressed genes involved in immune pathways from yellowhead catfish (Tachysurus fulvidraco) after poly (I:C) challenge.

Yellowhead catfish (Tachysurus fulvidraco) is an important aquaculture fish species in China with a high market value. Infectious diseases pose serious threats in farmed fish species, and although vaccines can prevent certain infections, they rely on potent adjuvants. In this study, we analyzed the transcriptomic profiles of spleens from poly (I:C)-treated T. fulvidraco. We obtained 46,362,922 reads corresponding to 490,926 transcripts and 318,059 genes. Gene annotation using different databases and subsequent differential gene expression analyses led to the identification of 5587 differentially expressed genes (DEGs), of which 2473 were up-regulated and 3114 were down-regulated in poly (I:C)-treated fish. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs revealed the significant dysregulation of immune- and cancer-related genes in the spleens of poly (I:C)-treated fish. Notably, several components of JAK-STAT, MAPK, and p53 signaling pathways were significantly dysregulated in response to poly (I:C) treatment. Quantitative real-time PCR (qRT-PCR) analysis of 11 randomly selected immune response genes confirmed the reliability of our findings. In conclusion, our findings provide novel insight into the immune responses of T. fulvidraco and suggest that poly (I:C) may represent a promising adjuvant of fish vaccines.

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.

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.

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.

A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) are conserved among insects and play an important role in the regulation of many biological processes, including temperature stress, abiotic stress, immune responses, metamorphosis, and embryo development. Antheraea pernyi is an economically valuable silk-producing moth and source of insect food containing high-quality protein. The aim of this study was to quantify expression of the ApsHSP21 gene in response to pathogen-associated molecular patterns (PAMPs) and nucleopolyhedrovirus (NPV) challenge. The deduced ApsHSP21 protein sequence consists of 186 residues with a calculated molecular mass of 21.0 kDa and an isoelectronic point (pI) of 6.63. The protein contains a conserved α-crystallin domain (ACD), and includes two casein kinase II phosphorylation sites, a protein kinase C phosphorylation site, two tyrosine kinase phosphorylation sites, and various polypeptide binding sites. Phylogenetic analysis revealed that ApsHSP21 is closely related to homologs from other insects. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that expression of ApsHSP21 was significantly up-regulated at different timepoints following simulated pathogen challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan, and NPV. The results suggest sHSP21 is involved in innate immune responses in A. pernyi.

A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge.

Ferritin plays important roles in iron storage, detoxification, and immune response. Here, a ferritin gene (PcFer) was identified in Procambarus clarkii, an economically important freshwater crayfish. Full-length PcFer cDNA was 1022-bp, including a 135-bp 5'-untranslated region (UTR) with a typical iron responsive element, a 374-bp 3'-UTR, and a 513-bp open reading frame encoding a polypeptide of 170 amino acids which contained the Ferritin domain. PcFer has ion binding sites, a ferrihydrite nucleation center, and an iron ion channel. PcFer is phylogenetically closely-related to Pacifastacus leniusculus and Eriocheir sinensis ferritins. Real-time quantitative reverse-transcription PCR analysis showed that PcFer was expressed in all tested P. clarkii tissues, and expressed most in hepatopancreas. After challenge with various heavy metals and lipopolysaccharide, respectively, the hepatopancreatic expression levels of PcFer were markedly upregulated. These results suggest that expression of PcFer might be involved in immune defense and protection of P. clarkii against heavy metal stress.

cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).

Hepcidin is a small, cysteine-rich antimicrobial peptide with a highly conserved ß-sheet structure that plays a vital role in innate host immunity against pathogenic organisms. In this study, a hepcidin gene was identified in Pelteobagrus fulvidraco, an economically important freshwater fish in China. The gene is named PfHep. The complete PfHep cDNA was 723 bp, including a 5'-untranslated region (UTR) of 102 bp, a 3'-UTR of 339 bp and an open reading frame of 282 bp encoding a polypeptide of 93 amino acids, which includes a predicted signal peptide and the Hepcidin domain. The predicted mature, cationic PfHep protein has a typical hepcidin RX (K/R)R motif and eight conserved cysteine residues. The deduced PfHep protein sequence has 70%, 54% and 39% percent identity with hepcidins from Ictalurus punctatus, Danio rerio, and Homo sapiens, respectively. The predicted tertiary structure of PfHep is very similar to that of hepcidin in other animals. Phylogenetic analysis revealed that PfHep is closely related to the hepcidins of I. punctatus and I. furcatus. Real-time quantitative reverse transcription-PCR showed that the PfHep gene was expressed most in liver of healthy P. fulvidraco, and expressed to some extent in all the tissues tested. After challenge with lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (poly I:C), respectively, the expression levels of PfHep were markedly upregulated in liver, spleen, head kidney and blood at different time points. Together these results imply that PfHep may be an important component of the innate immune system and be involved in immune defense against invading pathogens.

Molecular identification and expression analysis of a goose-type lysozyme (LysG) gene in yellow catfish Pelteobagrus fulvidraco.

Lysozymes, innate immunity molecules, play a vital role in immune response to pathogens. The yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. The aim of this study was to quantify expression of the P. fulvidraco LysG gene (a g-type lysozyme) in response to pathogen-associated molecular patterns (PAMP) challenge. First, the P. fulvidraco LysG gene (PfLysG) was cloned and characterized. The full-length cDNA of PfLysG is 1323 bp, including a 5'-untranslated region (UTR) of 131 bp, a 3'-UTR of 634 bp, and an open reading frame of 558 bp encoding a polypeptide of 185 amino acids, which contains a transglycosylase SLT domain (Pfam01464). The predicted molecular weight of the protein is 20.52 kDa with a pI of 9.08. Two catalytic residues and seven N-acetyl-D-glucosamine binding sites are conserved in the sequence and there is no predicted signal peptide. The deduced PfLysG protein sequence has 84%, 76% and 69% percent identity with the LysGs from Ictalurus furcatus, Danio rerio, and Salmo salar, respectively. The predicted tertiary structure of PfLysG is very similar to that from other animals. Phylogenetic analysis showed that PfLysG is closely related to those from Teleostei. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that PfLysG was expressed in all examined tissues and most highly expressed in head kidney, spleen, and intestine. After simulated pathogen challenge with lipopolysaccharide and polyriboinosinic polyribocytidylic acid, respectively, the mRNA expression of PfLysG was upregulated significantly at different time points. The results suggest that the identified g-type lysozyme of P. fulvidraco is involved in innate immune responses.

An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress.

The interaction of lysozyme with caffeine, theophylline and theobromine in solution.

The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV-Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)-lysozyme complex. The binding constants (K(A)) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.

Studies on the interaction between benzidine and hemocyanin from Chinese mitten crab Eriocheir japonica sinensis (Decapoda, Grapsidae).

The interaction of benzidine (BZ) with hemocyanin (Hc) from Chinese mitten crab (Eriocheir japonica sinensis) was studied by fluorescence spectrum. BZ can quench the fluorescence of Hc by forming a new complex. The process of binding BZ on Hc was a spontaneous molecular interaction procedure. The thermodynamic parameters, the enthalpy change and entropy change were estimated to be 47.36 kJ mol(-1), 248.51 J mol(-1) K(-1) according to the van' Hoff equation. This indicates that hydrophobic interaction played a major role in stabilizing the complex. The results of synchronous fluorescence spectroscopy and three-dimensional fluorescence spectra indicated that the structure of these tyrosine residues environments was altered by BZ, which could enter into the hydrophobic pocked of Hc.