Functional analyses of TRAF6 gene in Argopecten scallops.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

Whole-Genome Re-sequencing and Transcriptome Reveal Candidate Genes and Pathways Associated with Hybrid Sterility in Hermaphroditic Argopecten Scallops.

The interspecific hybrid scallops generated from the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) showed significant heterosis in growth. However, its sterility limits large-scale hybridization and hinders the development of the scallop breeding industry. Hybrid sterility is regulated by plenty of genes and involves a range of biochemical and physiological transformations. In this study, whole-genome re-sequencing and transcriptomic analysis were performed in sterile and fertile hybrid scallops. The potential genetic variations and abnormally expressed genes were detected to explore the mechanism underlying hybrid sterility in hermaphroditic Argopecten scallops. Compared with fertile hybrids, 24 differentially expressed genes (DEGs) with 246 variations were identified to be related to fertility regulation, which were mainly enriched in germarium-derived egg chamber formation, spermatogenesis, spermatid development, mismatch repair, mitotic and meiotic cell cycles, Wnt signaling pathway, MAPK signaling pathway, calcium modulating pathway, and notch signaling pathway. Specifically, variation and abnormal expression of these genes might inhibit the progress of mitosis and meiosis, promote cell apoptosis, and impede the genesis and maturation of gametes in sterile hybrid scallops. Eleven DEGs (XIAP, KAZN, CDC42, MEIS1, SETD1B, NOTCH2, TRPV5, M- EXO1, GGT1, SBDS, and TBCEL) were confirmed by qRT-PCR validation. Our findings may enrich the determination mechanism of hybrid sterility and provide new insights into the use of interspecific hybrids for extensive breeding.

Potential Involvement of DNA Methylation in Hybrid Sterility in Hermaphroditic Argopecten Scallops.

DNA methylation is an important epigenetic modification factor in regulating fertility. Corresponding process remains poorly investigated in hermaphroditic scallops. The interspecific F1 hybrids between the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) exhibited significant heterosis in yield, but sterility in hybrids obstructs the utilization of the genetic resources. However, the determination mechanism of hybrid sterility in the hermaphroditic Argopecten scallops is still unclear. In this study, the effect of DNA methylation in the hybrid sterility of hermaphroditic Argopecten scallops was explored. The results showed that the mean methylation level was higher in sterile hybrids than fertile hybrids, especially on chromosome 11 of the paternal parent. A total of 61,062 differentially methylated regions (DMRs) were identified, containing 3619 differentially methylated genes (DMGs) and 1165 differentially methylated promoters that are located in the DMRs of CG sequence context. The hyper-methylated genes were enriched into five KEGG pathways, including ubiquitin-mediated proteolysis, ECM-receptor interaction, non-homologous end-joining, notch signaling, and the mismatch repair pathways. The DMGs might induce hybrid sterility by inhibition of oogenesis and egg maturation, induction of apoptosis, increased ROS, and insufficient ATP supply. Our results would enrich the determination mechanism of hybrid sterility and provide new insights into the utilization of the genetic resources of the interspecific hybrids.

Potential roles of IFI44 genes in high resistance to Vibrio in hybrids of Argopecten scallops.

Vibrio bacteria are often fatal to aquatic organisms and selection of Vibrio-resistant strains is warranted for aquaculture animals. In this study, we found that hybrids between bay scallops and Peruvian scallops exhibited significantly higher resistance to Vibrio challenge, but little is available on its mechanism. Interferon induced protein 44 (IFI44), a member of the type I interferon (IFN) family, plays an important role in the IFN immune response in invertebrates, which may also participate in the resistance to Vibrio in scallops. To explore the roles of IFI44 genes in the resistance to Vibrio, they were identified and characterized in the bay scallop (designated as AiIFI44), the Peruvian scallop (designated as ApIFI44), and their reciprocal hybrids (designated as AipIFI44 and ApiIFI44, respectively). Their open reading frame (ORF) sequences were all 1434 bp, encoding 477 amino acids, but with large variations among the four genes. The AipIFI44 and ApiIFI44 exhibited higher similarity with ApIFI44 than with AiIFI44. All four genes have a TLDc structural domain with significant variations in sequences among them. Predicted differences in conformation and posttranslational modifications may lead to altered protein activity. We further demonstrated that the AiIFI44, AipIFI44 and ApiIFI44 expressed in all the tested tissues, with the highest expression in the gills and hepatopancreas. In response to Vibrio anguillarum challenge, the profile of mRNA expression of IFI44 gene differed among the bay scallops and the two hybrids. In the bay scallops, it increased at 6 h but dramatically decreased after 12-48 h. However, the mRNA expression of both AipIFI44 and ApiIFI44 decreased at 6 h but continuously increased thereafter and reached the highest value at 48 h. The results in the present study suggest the immune responds of IFI44 in scallops and it may be related to the higher resistance to Vibrio bacterial in hybrids.

Characterization of TRAF genes and their responses to Vibrio anguillarum challenge in Argopecten scallops.

The tumor necrosis factor receptor-related factor (TRAF) family has been reported to be involved in many immune pathways, such as TNFR, TLR, NLR, and RLR in animals. However, little is known about the roles of TRAF genes in the innate immune of Argopecten scallops. In this study, we first identified five TRAF genes, including TRAF2, TRAF3, TRAF4, TRAF6 and TRAF7, but not TRAF1 and TRAF5, from both the bay scallop A. irradians (Air) and the Peruvian scallop A. purpuratus (Apu). The phylogenetic analysis showed that the TRAF genes in Argopecten scallops (AiTRAF) belong to the branch of molluscan TRAF family, which lacks TRAF1 and TRAF5. Since TRAF6 is a key bridge factor in the tumor necrosis factor superfamily and plays an important role in innate and adaptive immunity, we cloned the ORFs of the TRAF6 gene in both A. irradians and A. purpuratus, as well as in two reciprocal hybrids (Aip for the hybrid Air × Apu and Api for the hybrid Apu × Air). Differences in conformational and post-translational modification resulted from the variation in amino acid sequences may cause differences in activity among them. Analysis of conserved motifs and protein structural domains revealed that AiTRAF contains typical structural domains similar to those of other mollusks and has the same conserved motifs. Tissue expression of TRAF in Argopecten scallops challenged by Vibrio anguillarum was examined by qRT-PCR. The results showed that AiTRAF were higher in gill and hepatopancreas. When challenged by Vibrio anguillarum, the expression of AiTRAF was significantly increased compared with the control group, indicating that AiTRAF may play an important role in the immunity of scallops. In addition, the expression of TRAF was higher in Api and Aip than in Air when challenged by Vibrio anguillarum, suggesting that TRAF may have contributed to the high resistance of Api and Aip to Vibrio anguillarum. The results of this study may provide new insights into the evolution and function of TRAF genes in bivalves and ultimately benefit scallop breeding.

Potential Roles of PTEN on Longevity in Two Closely Related Argopecten Scallops With Distinct Lifespans.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) has been found to regulate longevity through the PI3K/Akt/FoxO pathway and maintenance of genome integrity in worms, flies, and mammals. However, limited information is available on the roles of PTEN in longevity of aquatic animals. Here we extended this paradigm using two closely related Argopecten scallops, Argopecten purpuratus, and Argopecten irradians, with significantly distinct life spans, which are commercially important bivalve species for fishery and aquaculture in China, United States, Peru, and Chile. The ORFs of the ApPTEN and AiPTEN were 1,476 and 1,473 bp, which encoded 491 and 490 amino acids, respectively. There were 48 synonymous and 16 non-synonymous SNPs and one InDel of three nucleotides between ApPTEN and AiPTEN, resulting in variations in 15 amino acids and lack of S453 in AiPTEN. Differences in conformation and posttranslational modification were predicted between ApPTEN and AiPTEN, which may indicate different activities of ApPTEN and AiPTEN. When the animals were subjected to nutrition restriction, the expression of both ApPTEN and AiPTEN was upregulated, with AiPTEN responded faster and more robust than ApPTEN. Ionizing radiation induced significantly elevated expression of ApPTNE but not AiPTEN in the adductor muscle, and the mortality rate of A. purpuratus was significantly lower than that of A. irradians, indicating that ApPTNE may play a protective role by maintaining the genome integrity. RNAi of ApPTNE significantly downregulated the expression of its downstream regulated genes known to favor longevity, such as FoxO, Mn-SOD, and CAT. These results indicated that PTEN may contribute to the longevity of A. purpuratus through regulation of nutrient availability and genomic stability, probably via PI3K/Akt/FoxO pathway. Our study may provide new evidence for understanding of the conservative functions of PTEN in regulation of lifespan in animals and human, and it may also benefit the selection of scallops strains with long lifespan and thus larger size.

Mutations in Growth-Related Genes Induced by EMS Treatment in Scallops.

Background: The goal of genetic breeding is to select variants with mutations that are related to expected traits, such as fast growth. Artificial induction has been widely used to obtain strains with more mutations for further selection. Ethylmethylsulfone (EMS) is one of the most commonly used chemical mutagens in plant and microorganism breeding. However, the application of EMS mutagenesis in shellfish has not been reported. The aim of this study is to evaluate the potential use of EMS as a mutagen in scallop breeding, especially in characterization of mutations in growth-related genes. Results: Our results indicated that hatching of about 50% of fertilized eggs was blocked by treatment with 20 mM EMS for 3 h and the resulted larvae developed normally into adult stages. We then evaluated the mutagenic effects of EMS by sequencing the genomes of 4 adult scallops from the control group and 12 from the treatment group at 8 months after fertilization. On average, after removing shared types of mutations, there were 1,151,380 ± 258,188 SNPs (Single Nucleotide Polymorphisms) and 229,256 ± 51,714 InDels (insertion-deletion) in each animal in the EMS treatment group, while there were only134841 ± 10,115 SNPs and 42,605 ± 5,136 InDels in the control group. The average mutation rate in the genome of the EMS treatment group (0.0137 ± 0.0013%) was about 9 times that of the control group (0.0015 ± 0.0002%). GO (Gene Ontology) annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses revealed that mutations induced by EMS occurred evenly in most biological processes, cellular components and functions, as well in most pathways. However, significant lower percentage of mutations were found in the exonic region, in non-synonymous or Stopgain/Stoploss SNPs and in coding domains, suggesting apparent DNA repair or selection during grow-out stage. Analyses of the growth-related genes with mutations indicated that mutations in MFS (Major Facilitator Superfamily) and Tubulin were only found in the large-sized group (Five largest scallops: Treated-1, Treated-2, Treated-3, Treated-4, and Treated-5) and Homeobox and Socs (Suppressor of cytokine signaling) only in the small group (Two smallest scallops: Treated-11 and Treated-12). These results suggested that these genes may be involved in the regulation of growth in these animals, although further verification is certainly warranted. Conclusion: Treatment of fertilized eggs with 20 mM EMS for 3 h induced 9 times more mutations in scallop genomes. We found that mutations in MFS and Tubulin may be related to fast growth in the large-sized group and those mutations in Homeobox and SOCs may be involved in the slow growth in the small-sized scallops. EMS can be used to accelerate selection of economically important traits in molluscs.

Identification and functional analysis of a sex-biased transcriptional factor Foxl2 in the bay scallop Argopecten irradians irradians.

Transcription factor Foxl2 is an evolutionarily conserved gene playing pivotal roles in regulation of early ovarian differentiation and maintenance in animals. However, the Foxl2 gene has not been thoroughly studied in hermaphroditic scallops. In this study, we cloned and characterized a Foxl2 (designated as AiFoxl2) from the bay scallop Argopecten irradians irradians. The open reading frame of AiFoxl2 was 1122 bp encoding 373 amino acids residues and contained a conserved forkhead box domain. Quantitative real-time PCR showed that AiFoxl2 was mainly expressed in the ovary. Moreover, the highest expression of AiFoxl2 in the ovary was detected at proliferative stage and growing stage, while the lowest level was found at resting stage. During the embryonic and larval development, expression of AiFoxl2 was found first in fertilized eggs, increased significantly at the blastula stage, and reached peak value at the D-larvae stage. When AiFoxl2 was knocked down, testis development-related genes (Dmrt1, Sox7 and Sox9) were up-regulated significantly while the ovary development-related genes (Vg, HSD14, and GATA-1) were down-regulated manifestly. These findings suggested that AiFoxl2 was a female-related gene in A. i. irradians and may be involved in regulation of ovarian development and differentiation.

Transcriptomic analyses provide insights into the adaptive responses to heat stress in the ark shells, Scapharca subcrenata.

The ark shell, Scapharca subcrenata, is susceptible to high temperature which may lead to mass mortality in hot summers. Herein, we conducted the transcriptomic analyses of haemocytes in ark shells under thermal stress, to reveal the underlying molecular mechanisms of heat resistance in these animals. The results showed that a total of 7773, 11,500 and 13,046 unigenes were expressed differentially at 12, 24 and 48 h post thermal stress, respectively. The expression levels of key DEGs as revealed by RNA-seq were confirmed by quantitative real-time PCR. GO and KEGG enrichment analyses showed that the DEGs were mainly associated with apoptosis, NF-kappa B signaling pathway, TNF signaling pathway and RIG-I-like receptor signaling pathway. Among the DEGs, 40 were candidate heat stress response-related genes and 169 were identified to be involved in antioxidant defense, cell detoxification, protein metabolism and endoplasmic reticulum stress responses. It seemed that ark shells may adapt to short term thermal stress through regulation of protein metabolism, DNA replication and anti-apoptotic system. However, if the stress sustains, it may cause irreparable injury gradually in the animals due to oxygen limitation and metabolic dysregulation. Noteworthily, the expression of DEGs involved in protein biosynthesis and proteolysis was significantly elevated in ark shells under heat stress. These findings may provide preliminary insights into the molecular response of ark shells to acute thermal stress and lay the groundwork for marker-assisted selection of heat-resistant strains in S. subcrenata.

Transcriptomic analysis and expression of C-type lectins in response to Vibrio parahaemolyticus challenge in Scapharca subcrenata.

Little information is available on innate immune defense mechanisms of Scapharca subcrenata. C-type lectins (CTLs) are not only pattern recognition proteins that can bind pathogen-associated molecular patterns, but also crucial maternally-derived immune factors in mollusc egg. In this study, the comparative transcriptome analysis of Vibrio parahaemolyticus-infected and untreated hepatopancreas were performed to identify the key genes involved in maternal transfer of immunity. A total of 3514 and 9327 differentially expressed genes (DEGs) were identified at 6 and 48 h post challenge compared to control groups. Gene Ontology and Cluster of Orthologous Groups analysis showed that most DEGs were classified under regulation of signal transduction, regulation of the metabolic process of carbohydrates and secondary metabolites, while the processes of posttranscriptional modification and protein translation were inhibited manifestly. The DEGs were most enriched in pathways related to lysosome, phagosome and EMC-receptor interaction. Among the DEGs, 191 maternal immune-related genes that could provide developing embryos a better protection against pathogen infection were identified according to previous studies. Additionally, five CTLs (designated as SsCTL1-5) identified from the DEGs were cloned, and their expression patterns in different tissues and post immune stimulation were analyzed. These findings would be beneficial for understanding the innate immune defense mechanisms of S. subcrenata.

Draft genomes of two Atlantic bay scallop subspecies Argopecten irradians irradians and A. i. concentricus.

The two subspecies of Atlantic bay scallop (Argopecten irradians), A. i. irradians and A. i. concentricus, are economically important aquacultural species in northern and southern China. Here, we performed the whole-genome sequencing, assembly, and gene annotation and produced draft genomes for both subspecies. In total, 253.17 and 272.97 gigabases (Gb) of raw reads were generated from Illumina Hiseq and PacBio platforms for A. i. irradians and A. i. concentricus, respectively. Draft genomes of 835.7 Mb and 874.82 Mb were assembled for the two subspecies, accounting for 83.9% and 89.79% of the estimated sizes of their corresponding genomes, respectively. The contig N50 and scaffold N50 were 78.54 kb and 1.53 Mb for the A. i. irradians genome, and those for the A. i. concentricus genome were 63.73 kb and 1.25 Mb. Moreover, 26,777 and 25,979 protein-coding genes were predicted for A. i. irradians and A. i. concentricus, respectively. These valuable genome assemblies lay a solid foundation for future theoretical studies and provide guidance for practical scallop breeding.

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.

Characterization and functional analysis of a novel mannose-binding lectin from the swimming crab Portunus trituberculatus.

Mannose-binding lectin (MBL) is a pattern recognition receptor (PRR) that plays an important role in the innate immune response. In this study, a novel mannose-binding lectin was cloned from the swimmimg crab Portunus trituberculatus (designated as PtMBL). The complete cDNA of PtMBL gene was 1208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gill with sexually dimorphic expression. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca2+-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d-galactose and d-mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. These results together indicate that PtMBL could serve as not only a PRR in immune recognition but also a potential antibacterial protein in the innate immune response of crab.

Two alpha-2 macroglobulin from Portunus trituberculatus involved in the prophenoloxidase system, phagocytosis and regulation of antimicrobial peptides.

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes.

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.