Min Hui

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.

Comparative Transcriptome Analysis Reveals Sex-Biased Gene Expression in Juvenile Chinese Mitten Crab Eriocheir sinensis

Sex-biased genes are considered to account for most of phenotypic differences between males and females. In order to explore the sex-biased gene expression in crab, we performed the whole-body transcriptome analysis in male and female juveniles of the Chinese mitten crab Eriocheir sinensis using next-generation sequencing technology. Of the 23,349 annotated unigenes, 148 were identified as sex-related genes. A total of 29 candidate genes involved in primary sex determination pathways were detected, indicating the sex determination cascade of the mitten crab might be more complex than previously supposed. Differential expression analysis showed 448 differentially expressed genes (DEGs) between the two transcriptomes. Most of DEGs were involved in processes such as metabolism and immunity, and not associated with obvious sexual function. The pathway predominantly enriched for DEGs were related to lysosome, which might reflect the differences in metabolism between males and females. Of the immune DGEs, 18 up-regulated genes in females were humoral immune factors, and eight up-regulated genes in males were pattern recognition receptors, suggesting sex differences of immune defense might exist in the mitten crab. In addition, two reproduction-related genes, vitellogenin and insulin-like androgenic gland factor, were identified to express in both sexes but with significantly higher level in males. Our research provides the first whole-body RNA sequencing of sex-specific transcriptomes for juvenile E. sinensis and will facilitate further studies on molecular mechanisms of crab sexual dimorphism.

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.

Comparative transcriptomic analysis provides insights into the molecular basis of brachyurization and adaptation to benthic lifestyle in Eriocheir sinensis.

The development of Eriocheir sinensis from the megalopa to the first juvenile crab undergoes brachyurization and a habitat transition from pelagic to benthic lifestyle. However, the molecular mechanism of this process is poorly understood in crabs. Using next-generation RNA sequencing, we identified 22,622 annotated unigenes and 1016 differentially expressed genes especially involved in metamorphosis, sensory perception and immunity in the two stages. Among 22,622 unigenes, 169 unigenes were assigned to morphogenic pathways (i.e., Wnt, Hedgehog and hormone biosynthesis). For metamorphosis, 38 up-regulated genes in megalopae were associated with cytoskeleton construction, while, 52 up-regulated genes in juvenile were related to cuticle development and protein degradation. Several crucial genes involved in phototransduction and two olfactory-related genes were significantly up-regulated in megalopae. Moreover, 44 immunity-related genes were highly expressed in megalopae. This study provides a comprehensive view of brachyurization and benthic adaptation mechanisms in E. sinensis.

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.

Molecular characterization and expression profile of three Fem-1 genes in Eriocheir sinensis provide a new insight into crab sex-determining mechanism.

The FEM-1 protein of Caenorhabditis elegans plays a crucial role in the nematode sex-determination pathway. Here, we reported the characterization of three members of Fem-1 gene family in Eriocheir sinensis (designated EsFem-1a, EsFem-1b, and EsFem-1c), which were homologs of the nematode FEM-1 protein. The amino acid sequences of EsFem-1a, EsFem-1b, and EsFem-1c contained eight, nine, and eight ankyrin repeats, respectively. None of the ankyrin repeats had its own specific signature, and the evolution of ankyrin repeat was not completely independent. The predicted three-dimensional structure of EsFem-1 proteins exhibited highly similar superhelical conformation, especially the N-terminal six contiguous ankyrin repeats, which provided a binding surface for the protein-protein interaction. Phylogenetic tree based on the amino acid sequences revealed that EsFem-1a, EsFem-1b, and EsFem-1c were divided into three obvious separated clades. EsFem-1 genes were highly expressed in fertilized egg, 2-4 cell and blastula stage comparing with larval stage (P<0.01), which suggested they might be maternal genes. They also showed a certain degree of sexually dimorphic expression in some tissues. Notably, the highest expression of EsFem-1a was in the hepatopancreas, with EsFem-1b in testes and EsFem-1c in muscle (P<0.05), which indicated their potential role in a broad array of tissues. In addition, the genes initially involved in sex differentiation were not limited to those specifically expressed in the developing gonad. Taken together, these results suggested that EsFem-1 might function in crab early sex determination and late gonad development. The identification of Fem-1 gene family in E. sinensis provides a new insight into crab sex-determination mechanism.

First complete mitochondrial genome of primitive crab Homologenus malayensis (Decapoda: Brachyura: Podotremata: Homolidae).

Abstract The taxonomy and phylogeny of brachyuran crabs, and particularly of the Podotremata, have been the subjects of controversy due to their morphological diversity and complexity. The first complete mitochondrial genome (mitogenome) sequence of primitive crab Homologenus malayensis has been determined. The mitogenome is 15,793 bp in length, with A + T content 71.7%. The gene content and order are consistent with those in typical brachyuran crabs. A putative control region of 883 bp is identified due to its position (between srRNA and tRNAIle) and AT richness (75.5%). Notably, the control region in H. malayensis contains nine identical specific repeat units of 42 bp and 11 identical repeat units of 2 bp with a total length of 400 bp, which is different from other crabs. These results are expected to provide useful information on both genomics and the future phylogenetic study of primitive crabs.

PtSerpin from the swimming crab Portunus trituberculatus, a putative regulator of prophenoloxidase activation with antibacterial activity

Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 μM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system.