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.

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.

Molecular identification and expression analysis of natural resistance-associated macrophage protein (Nramp) gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).

Natural resistance associated macrophage protein genes (Nramp) is one of the important candidate genes responsible for regulating immune response against pathogen infection. The aim of the present was to quantify expression of Nramp gene in response to pathogen infection. Here, a Nramp was identified and molecularly characterized from Pelteobagrus fulvidraco (PfNramp). The obtained 3134 bp cDNA fragment of PfNramp comprised a 5'-untranslated region (UTR) of 81 bp, a 3'-UTR of 1403 bp and an open reading frame (ORF) of 1650 bp, encoding a polypeptide of 549 amino acids that contained a typical structural features of Nramp domain (Pfam01566). BLAST analysis exhibited that PfNramp shared sequence similarity to other organisms, in particular to Ictalurus furcatus (92%), Danio rerio (82%), and Homo sapiens (77%). Phylogenetic analysis revealed that PfNramp is close to Teleostei. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that PfNramp was expressed in all examined tissues, with the highest abundance in liver. The mRNA expression of PfNramp was remarkably increased at different time points after lipopolysaccharide (LPS), and polyriboinosinic polyribocytidylic acid (poly I:C) challenge. These results suggest that PfNramp is an inducible protein in the innate immune reactions of P. fulvidraco and probably in other fish species.

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.

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.