Guohui Shi

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Hepatopancreas of Microbial Challenged Mitten Crab Eriocheir sinensis

Background The Chinese mitten crab Eriocheir sinensis is an important economic crustacean and has been seriously attacked by various diseases, which requires more and more information for immune relevant genes on genome background. Recently, high-throughput RNA sequencing (RNA-seq) technology provides a powerful and efficient method for transcript analysis and immune gene discovery. Methods/Principal Findings A cDNA library from hepatopancreas of E. sinensis challenged by a mixture of three pathogen strains (Gram-positive bacteria Micrococcus luteus, Gram-negative bacteria Vibrio alginolyticus and fungi Pichia pastoris; 108 cfu·mL−1) was constructed and randomly sequenced using Illumina technique. Totally 39.76 million clean reads were assembled to 70,300 unigenes. After ruling out short-length and low-quality sequences, 52,074 non-redundant unigenes were compared to public databases for homology searching and 17,617 of them showed high similarity to sequences in NCBI non-redundant protein (Nr) database. For function classification and pathway assignment, 18,734 (36.00%) unigenes were categorized to three Gene Ontology (GO) categories, 12,243 (23.51%) were classified to 25 Clusters of Orthologous Groups (COG), and 8,983 (17.25%) were assigned to six Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Potentially, 24, 14, 47 and 132 unigenes were characterized to be involved in Toll, IMD, JAK-STAT and MAPK pathways, respectively. Conclusions/Significance This is the first systematical transcriptome analysis of components relating to innate immune pathways in E. sinensis. Functional genes and putative pathways identified here will contribute to better understand immune system and prevent various diseases in crab.

High-density linkage mapping aided by transcriptomics documents ZW sex determination system in the Chinese mitten crab Eriocheir sinensis

The sex determination system in crabs is believed to be XY-XX from karyotypy, but centromeres could not be identified in some chromosomes and their morphology is not completely clear. Using quantitative trait locus mapping of the gender phenotype, we revealed a ZW-ZZ sex determination system in Eriocheir sinensis and presented a high-density linkage map covering ~98.5% of the genome, with 73 linkage groups corresponding to the haploid chromosome number. All sex-linked markers in the family we used were located on a single linkage group, LG60, and sex linkage was confirmed by genome-wide association studies (GWAS). Forty-six markers detected by GWAS were heterozygous and segregated only in the female parent. The female LG60 was thus the putative W chromosome, with the homologous male LG60 as the Z chromosome. The putative Z and W sex chromosomes were identical in size and carried many homologous loci. Sex ratio (5:1) skewing towards females in induced triploids using unrelated animals also supported a ZW-ZZ system. Transcriptome data were used to search for candidate sex-determining loci, but only one LG60 gene was identified as an ankyrin-2 gene. Double sex- and mab3-related transcription factor 1 (Dmrt1), a Z-linked gene in birds, was located on a putative autosome. With complete genome sequencing and transcriptomic data, more genes on putative sex chromosomes will be characterised, thus leading towards a comprehensive understanding of the sex determination and differentiation mechanisms of E. sinensis, and decapod crustaceans in general.

Transcriptome changes in Eriocheir sinensis megalopae after desalination provide insights into osmoregulation and stress adaption in larvae.

Eriocheir sinensis, an extremely invasive alien crab species, has important economic value in China. It encounters different salinities during its life cycle, and at the megalopal stage it faces a turning point regarding the salinity in its environment. We applied RNA sequencing to E. sinensis megalopae before (MB) and after (MA) desalination, resulting in the discovery of 21,042 unigenes and 908 differentially expressed genes (DEGs, 4.32% of the unigenes). The DEGs primarily belonged to the Gene Ontology groups “Energy metabolism,” “Oxidoreductase activity,” “Translation,” “Transport,” “Metabolism,” and “Stress response.” In total, 33 DEGs related to transport processes were found, including 12 proton pump genes, three ATP-binding cassettes (ABCs), 13 solute carrier (SLC) family members, two sweet sugar transporter (ST) family members and three other substance transporters. Mitochondrial genes as well as genes involved in the tricarboxylic acid cycle, glycolytic pathway, or β-oxidation pathway, which can generate energy in the form of ATP, were typically up-regulated in MA. 11 unigenes related to amino acid metabolism and a large number of genes related to protein synthesis were differentially expressed in MB and MA, indicating that E. sinensis possibly adjusts its concentration of free amino acid osmolytes for hyper-osmoregulation. Additionally, 33 salinity and oxidative stress induced genes were found to be differentially expressed, such as the LEA2, HSPs, GST and coagulation factor genes. Notably, LEA2 is an extremely hydrophilic protein that responds to desiccation and reported for the first time in crabs. Therefore, we suppose that when the environment is hypo-osmotic, the megalopae might compensate for ion loss via hyper-osmoregulation by consuming more energy, accompanied by a series of stress induced adaptions. This study provides the first genome-wide transcriptome analysis of E. sinensis megalopae for studying its osmoregulation and stress adaption mechanisms.

Transcriptome Profiling Analysis on Whole Bodies of Microbial Challenged Eriocheir sinensis Larvae for Immune Gene Identification and SNP Development

To study crab immunogenetics of individuals, newly hatched Eriocheir sinensis larvae were stimulated with a mixture of three pathogen strains (Gram-positive bacteria Micrococcus luteus, Gram-negative bacteria Vibrio alginolyticus and fungi Pichia pastoris; 108 cfu·mL-1). A total of 44,767,566 Illumina clean reads corresponding to 4.52 Gb nucleotides were generated and assembled into 100,252 unigenes (average length: 1,042 bp; range: 201-19,357 bp). 17,097 (26.09%) of 65,535 non-redundant unigenes were annotated in NCBI non-redundant protein (Nr) database. Moreover, 23,188 (35.38%) unigenes were assigned to three Gene Ontology (GO) categories, 15,071 (23.00%) to twenty-six Clusters of orthologous Groups (COG) and 8,574 (13.08%) to six Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Numerous genes were further identified to be associated with multiple immune pathways, including Toll, immune deficiency (IMD), janus kinase (JAK)-signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Some of them, such as tumor necrosis factor receptor associated factor 6 (TRAF6), fibroblast growth factor (FGF), protein-tyrosine phosphatase (PTP), JNK-interacting protein 1 (JIP1), were first identified in E. sinensis. TRAF6 was even first discovered in crabs. Additionally, 49,555 single nucleotide polymorphisms (SNPs) were developed from over 13,309 unigenes. This is the first transcriptome report of whole bodies of E. sinensis larvae after immune challenge. Data generated here not only provide detail information to identify novel genes in genome reference-free E. sinensis, but also facilitate our understanding on host immunity and defense mechanism of the crab at whole transcriptome level.

Molecular cloning, genomic structure and antimicrobial activity of PtALF7, a unique isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus

Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, are becoming predominant candidates for potential therapeutic agents of bacterial and viral diseases. In this study, a unique isoform of ALF (PtALF7) was identified from hemocytes cDNA library of the swimming crab Portunus trituberculatus. The PtALF7 cDNA contained an open reading frame (ORF) of 372 bp encoding 123 amino acids. The deduced peptide of PtALF7 shared high similarity with our previously reported PtALF1-3 but low with PtALF4-6. The PtALF7 gene consisted of three exons interrupted by two introns, and was clearly transcribed from different genomic loci compared with other PtALF isoforms. Totally 128 SNPs including 12 in coding region and 116 in noncoding region were detected in PtALF7 gene by direct sequencing of 20 samples. The mRNA expression of PtALF7 transcript was primarily observed in hemocytes followed by gill and eyestalk, but barely detectable in hepatopancreas. After challenge with Vibrio alginolyticus, a main pathogen causing high mortality in P. trituberculatus, the PtALF7 transcript in hemocytes showed a clear time-dependent response expression pattern with obvious decrease at 6 h and significant increase at 24 h. The recombinant PtALF7 protein exhibited antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate a potential involvement for PtALF7 in the innate immune response of P. trituberculatus.

A newly identified anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus with broad spectrum antimicrobial activity

Anti-lipopolysaccharide factors (ALFs), exhibiting binding and neutralizing activities to lipopolysaccharide (LPS), are the potent antimicrobial peptides of innate immunity in crustaceans. In this study, a unique isoform of ALF (PtALF6) was identified from eyestalk cDNA library of the swimming crab Portunus trituberculatus. The full-length cDNA of PtALF6 was 669 bp encoding 115 amino acids, relatively short to other known ALFs. The deduced peptide of PtALF6 was conserved; it contained the signal peptide and LPS-binding domain, especially the two conserved cysteine residues at both ends of the domain. Predicted tertiary structures of PtALF6 containing four β-strands and three α-helices were similar to that described in Limulus polyphemus. The genomic fragment of PtALF6 contained three exons separated by two introns. Unlike most ALFs expressed in hemocytes, PtALF6 transcript was predominantly detected in gill with 14.05-fold higher than that in hemocytes. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF6 transcript in hemocytes showed a clear time-dependent response expression pattern with two significant peaks at 12 h and 32 h post-injection. The recombinant PtALF6 protein revealed antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate that PtALF6 is a potential antimicrobial protein against Gram-negative and Gram-positive bacteria infection, and may play an important role in innate immune response of P. trituberculatus.

Unusual sequence features and gene rearrangements of primitive crabs revealed by three complete mitochondrial genomes of Dromiacea.

Three complete mitochondrial genomes of primitive crabs, Dynomene pilumnoides (belong to Dromioidea), Homola orientalis and Moloha majora (belong to Homoloidea) were determined, characterized and compared with other brachyuran crabs. Due to the presence of four intergenic noncoding sequences (IGNs), the complete length of 16,475bp in D. pilumnoides is relatively large among brachyuran mitogenomes. Its longest IGN is 652bp in size locating between trnL1 and trnQ, which is regarded as putative control region (CR) considering its high similarity with the same superfamily species, Conchoecetes artificiosus and Takedromia cristatipes. Compared with the gene order of putative ancestors of insects and crustaceans, D. pilumnoides mitogenome exhibits rearrangements with positional translocation of three genes (trnQ, CR and trnH). The mitogenomes of H. orientalis and M. majora are 16,084bp and 15,903bp in size, respectively. Their gene arrangements are consistent with those reported for most brachyuran species. The lengths of CR in these two Homoloidea crabs are 1242bp and 1037bp, respectively. The occurrence of tandem repeat sequences (TRS) in control region is shared by the three Homoloidea crabs (besides Homologenus malayens), but is not found in the other reported brachyuran crabs. Moreover, the repeat units in H. orientalis and H. malayens show the high level of sequence identity with stable secondary structure. The phylogenetic analyses indicate that Dromioidea and Homoloidea form the most basal assemblage within Brachyura, followed by Raninoidea.

The complete mitochondrial genomes of Umalia orientalis and Lyreidus brevifrons: The phylogenetic position of the family Raninidae within Brachyuran crabs

The complete mitochondrial genome (mitogenome) sequences of two primitive crabs, Umalia orientalis and Lyreidus brevifrons (Decapoda: Brachyura: Raninidae) were determined. The mitogenomes of the two species are 15,466 and 16,112bp in length with AT content of 68.0% and 70.6%, respectively. Each genome contains 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes. The gene arrangement of U. orientalis is the same with those reported for most brachyuran species. Nevertheless, the gene arrangement of L. brevifrons differs from that of U. orientalis in having an additional non-coding region. The newly found non-coding region is located between nad3 and trnA with 641bp in length. Its nucleotide composition and secondary structure are similar to the typical control region. In L. brevifrons, the secondary structures of trnS-AGN and trnI are significantly different from those in U. orientalis and other brachyuran species. The start codon for cox1 is ATG in all reported Eubrachyura mitogenomes, while a common start codon ACG is found in the Podotremata. Phylogenetic analyses for crustacean decapods based on the nucleotide and amino acid of 13 PCGs indicate that Homolidae is more primitive in Brachyura, and Raninidae is a sister group to Eubrachyura. This implies that Raninidae is closer to Eubrachyura than to Homolidae, and Podotremata may be a paraphyletic assemblage. The results also indicate that the subfamily Lyreidinae is closer to Notopodinae than to Ranininae within Raninidae. The novel mitogenome data provides useful information for refining the phylogenetic relationships within Brachyura.

Characterization and functional analysis of serine proteinase and serine proteinase homologue from the swimming crab Portunus trituberculatus

Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity.

PtPLC, a pacifastin-related inhibitor involved in antibacterial defense and prophenoloxidase cascade of the swimming crab Portunus trituberculatus

Pacifastin-related inhibitor is a new family of serine protease inhibitors that regulate the proteolytic cascade in multiple biological processes. Contrary to the knowledge on the structure and inhibitory mechanism of pacifastin-like members in locust, very little is known about their functions. Here, we report the inhibitory activities in relation to the structural characteristics of pacifastin light chain (PtPLC) gene identified from the swimming crab Portunus trituberculatus. The mature PtPLC and five PLD-related domains with critical residues were expressed in Escherichia coli, and assayed for their activities. The recombinant PtPLC (rPtPLC) displayed inhibitory activities against trypsin and chymotrypsin in a dose dependent manner, with a preference for trypsin. Except for rPtPLC-D4, the other four rPtPLC-related domains could inhibit at least one of serine proteases. The enzyme specificity of PtPLC domains generally corresponded to the nature of the P1 residue at the reactive site. rPtPLC was able to inhibit the growth of Gram-negative bacteria Vibrio alginolyticus and Pseudomonas aeruginosa, but not the Gram-positive bacterium and fungus tested. Further phenoloxidase (PO) assay showed the rPtPLC could depress the crab proPO system activation in vitro, and lead to 72.8% inhibition of PO activity at the concentration of 9.11 μM. It also suppressed proPO activation induced by rPtcSP and rPtSPH1. As the first functional study of the recombinant PLC protein in crustaceans, the present results together indicate that PtPLC functions in the crab immune response possibly via inhibiting bacterial growth and regulating the proPO system.