The Chinese mitten crab genome provides insights into adaptive plasticity and developmental regulation.

The infraorder Brachyura (true or short-tailed crabs) represents a successful group of marine invertebrates yet with limited genomic resources. Here we report a chromosome-anchored reference genome and transcriptomes of the Chinese mitten crab Eriocheir sinensis, a catadromous crab and invasive species with wide environmental tolerance, strong osmoregulatory capacity and high fertility. We show the expansion of specific gene families in the crab, including F-ATPase, which enhances our knowledge on the adaptive plasticity of this successful invasive species. Our analysis of spatio-temporal transcriptomes and the genome of E. sinensis and other decapods shows that brachyurization development is associated with down-regulation of Hox genes at the megalopa stage when tail shortening occurs. A better understanding of the molecular mechanism regulating sexual development is achieved by integrated analysis of multiple omics. These genomic resources significantly expand the gene repertoire of Brachyura, and provide insights into the biology of this group, and Crustacea in general.

Identification, functional characterization and the potential role of variable lymphocyte receptor EsVLRA from Eriocheir sinensis in response to secondary challenge after Vibrio parahaemolyticus vaccine.

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

Functional characterization of two clip-domain serine proteases in the swimming crab Portunus trituberculatus.

Clip domain serine proteases (cSPs), a family of multifunctional proteins, play a crucial role in innate immune system. Here, we report the functional characterization of two clip domain serine proteases (PtcSP1 and PtcSP3) from the swimming crab Portunus trituberculatus. The recombinant N-terminal clip domains and the C-terminal SP-like domains of PtcSP1 and PtcSP3 were expressed in Escherichia coli system, and assayed for various biological functions: protease activity, antimicrobial activity, bacterial clearance and microbial-binding activity. The recombinant SP-like domains of PtcSP1 and PtcSP3 exhibited trypsin-like protease activity, while their recombinant clip domains showed strong antibacterial activity and could bind to bacteria and yeast, suggesting the potential roles of PtcSP1 and PtcSP3 in immune defense and pattern recognition. Unlike PtcSP3, PtcSP1 revealed the opsonic activity as shown by a higher bacterial clearance rate of Vibrio alginolyticus coated with the combination of the recombinant clip domain and SP-like domain of PtcSP1 as compared with V. alginolyticus only. Knockdown of PtcSP1 or PtcSP3 by RNA interference resulted in a significant decrease of total phenoloxidase (PO) activity in crab, suggesting that PtcSP1 and PtcSP3 are involved in the proPO system. In addition, suppression of PtcSP1 or PtcSP3 changed the expression of PtALFs and complement-like components. All these findings suggest that PtcSP1 and PtcSP3 are multifunctional immune molecules and perform different protective functions in crab defense.

Comparative transcriptome analysis explores maternal to zygotic transition during Eriocheir sinensis early embryogenesis.

The maternal genome directs almost all aspects of early animal development. As development proceeds, the elimination of maternal gene products and zygotic genome activation (ZGA) occur during the maternal to zygotic transition (MZT). To study the molecular mechanisms regulating this developmental event in Eriocheir sinensis, RNA-Seq technology was applied to generate comprehensive information on transcriptome dynamics during early embryonic stages. In total, 32,088 annotated unigenes were obtained from the transcriptomes of fertilized eggs and embryos at the cleavage (2-4 cell) and blastula stage. A total of 566 maternal genes and 1165 zygotic genes were isolated, among which 103 and 266 genes were predicted conserved maternal transcripts (COMATs) and conserved zygotic transcripts (COZYTs), respectively. The COMATs performed housekeeping gene functions and may be essential for initiating early embryogenesis of the Bilateria. Furthermore, 87, 76 and 117 differentially expressed genes associated with the MZT, morphogenesis and immunity were identified when compared the three transcriptomic datasets. We also unmask that the MZT takes place around the cleavage stage, when the genes involved in the clearance of maternal gene products and the ZGA were significantly up-regulated. Taken together, these datasets provide a valuable resource for understanding the mechanisms of early developmental events in E. sinensis, and facilitate further studies on molecular mechanisms of asynchronous development in crabs.

Characterization and functional analysis of a novel gC1qR in the swimming crab Portunus trituberculatus.

The receptor for the globular head of complement component C1q, gC1qR, is a multifunctional and multiligand binding protein with a crucial role in host defense. In the present study, a full-length cDNA sequence of a gC1qR homolog (PtgC1qR) in Portunus trituberculatus was identified. PtgC1qR was a 268-amino-acid polypeptide with a conserved MAM33 domain and a mitochondrial targeting sequence in the first 56 amino acids. The transcripts of PtgC1qR were detected in all examined tissues with the highest level detected in the hepatopancreas. Compared with other early embryonic stages, PtgC1qR was highly expressed in the fertilized eggs and embryos at the cleavage stage, which suggest PtgC1qR may be a maternal gene. The transcripts of PtgC1qR in hemocytes exhibited time-dependent response expression pattern after challenged with bacteria (Vibrio alginolyticus, Micrococcus luteus) and fungi (Pichia pastoris). Moreover, the recombinant PtgC1qR (rPtgC1qR) exhibited strong antibacterial activity and microbial-binding activity, suggesting its crucial role in immune defense and recognition. Further phenoloxidase (PO) assay showed that rPtgC1qR could suppress the crab PO activity in vitro in a dose-dependent manner, and it could result in nearly 100% inhibition of PO activity under the concentration of 11.65 µM. Knockdown of PtgC1qR could significantly enhance the expression of serine protease related genes (PtSP1-3 and PtSPH), proPO-associated genes (PtproPO and PtPPAF) and C3-like genes (Ptα2M1 and PtTEP). However, the phagocytosis related genes (PtMyosin, PtRab5 and PtArp) and Ptα2M2 were significantly down-regulated in the PtgC1qR silenced crabs. These findings together demonstrate that PtgC1qR might function in crab immune response via its antibacterial activity, immune recognition or regulating the proPO system, complement pathway and phagocytosis.

A chymotrypsin-like serine protease from Portunus trituberculatus involved in pathogen recognition and AMP synthesis but not required for prophenoloxidase activation.

Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus. The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression.