Cloning and functionally characterization of TtVtg2-like in horseshoe crab Tachypleus tridentatus: A special focus on ovarian development.

Horseshoe crabs are living fossils. In recent decades, the population of horseshoe crabs, especially the tri-spine horseshoe crab Tachypleus tridentatus, has decreased significantly and was listed as an 'endangered species' under the IUCN Red List in 2019. In order to improve the reproduction of T. tridentatus to facilitate stock enhancement, it is important to understand their ovarian development. In this study, a novel TtVtg2-like gene from T. tridentatus was cloned and functionally characterized. The total legth of TtVtg2-like was 5469 bp, encoding a protein consisting of 1822 amino acid with a pI value of 6.51 and a molecular weight of 208.68 KDa. The TtVtg2-like was highly expressed in the ovary and yellow connective tissues, mainly localized in cytoplasm and endoplasmic reticulum vesicles of oocytes and yellow connective tissues, respectively. RNA interference of TtVtg2-like caused the accumulation of ROS, DNA damage, and apoptosis of ovarian primary cells. The results of this study provide useful baseline information for future studies on ovarian development in horseshoe crabs.

Lower Ordovician synziphosurine reveals early euchelicerate diversity and evolution.

Euchelicerata is a clade of arthropods comprising horseshoe crabs, scorpions, spiders, mites and ticks, as well as the extinct eurypterids (sea scorpions) and chasmataspidids. The understanding of the ground plans and relationships between these crown-group euchelicerates has benefited from the discovery of numerous fossils. However, little is known regarding the origin and early evolution of the euchelicerate body plan because the relationships between their Cambrian sister taxa and synziphosurines, a group of Silurian to Carboniferous stem euchelicerates with chelicerae and an unfused opisthosoma, remain poorly understood owing to the scarce fossil record of appendages. Here we describe a synziphosurine from the Lower Ordovician (ca. 478 Ma) Fezouata Shale of Morocco. This species possesses five biramous appendages with stenopodous exopods bearing setae in the prosoma and a fully expressed first tergite in the opisthosoma illuminating the ancestral anatomy of the group. Phylogenetic analyses recover this fossil as a member of the stem euchelicerate family Offacolidae, which is characterized by biramous prosomal appendages. Moreover, it also shares anatomical features with the Cambrian euarthropod Habelia optata, filling the anatomical gap between euchelicerates and Cambrian stem taxa, while also contributing to our understanding of the evolution of euchelicerate uniramous prosomal appendages and tagmosis.

Targeting TtVgR via siRNA Knockdown Elicits Ovarian Cell Death in the Tri-spine Horseshoe Crab.

The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.

Impact of hypoxia on glucose metabolism and hypoxia signaling pathways in juvenile horseshoe crabs Tachypleus tridentatus.

Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.

Combined effects of norfloxacin and polystyrene nanoparticles on the oxidative stress and gut health of the juvenile horseshoe crab Tachypleus tridentatus.

Pollution with anthropogenic contaminants including antibiotics and nanoplastics leads to gradual deterioration of the marine environment, which threatens endangered species such as the horseshoe crab Tachypleus tridentatus. We assessed the potential toxic mechanisms of an antibiotic (norfloxacin, 0, 0.5, 5 µg/L) and polystyrene nanoparticles (104 particles/L) in T. tridentatus using biomarkers of tissue redox status, molting, and gut microbiota. Exposure to single and combined pollutants led to disturbance of redox balance during short-term (7 days) exposure indicated by elevated level of a lipid peroxidation product, malondialdehyde (MDA). After prolonged (14-21 days) exposure, compensatory upregulation of antioxidants (catalase and glutathione but not superoxide dismutase) was observed, and MDA levels returned to the baseline in most experimental exposures. Transcript levels of molting-related genes (ecdysone receptor, retinoic acid X alpha receptor and calmodulin A) and a molecular chaperone (cognate heat shock protein 70) showed weak evidence of response to polystyrene nanoparticles and norfloxacin. The gut microbiota T. tridentatus was altered by exposures to norfloxacin and polystyrene nanoparticles shown by elevated relative abundance of Bacteroidetes. At the functional level, evidence of suppression by norfloxacin and polystyrene nanoparticles was found in multiple intestinal microbiome pathways related to the genetic information processing, metabolism, organismal systems, and environmental information processing. Future studies are needed to assess the physiological and health consequences of microbiome dysbiosis caused by norfloxacin and polystyrene nanoparticles and assist the environmental risk assessment of these pollutants in the wild populations of the horseshoe crabs.

Integrated transcriptomics and metabolomics reveal the toxic mechanisms of mercury exposure to an endangered species Tachypleus tridentatus.

Mercury (Hg) pollution is threatening the health of endangered Tachypleus tridentatus whereas the toxic mechanism is still unclear. This study combined transcriptomic and metabolomics technology to reveal the toxic mechanisms of mercury (Hg 2+, 0.025 mg/L) exposing to T. tridentatus larvae for 15 days. Mercury induced cellular toxicity and cardiovascular dysfunction by dysregulating the genes related to endocrine system, such as polyubiquitin-A, cathepsin B, atrial natriuretic peptide, etc. Mercury induced lipid metabolic disorder with the abnormal increase of lysoPC, leukotriene D4, and prostaglandin E2. Cytochrome P450 pathway was activated to produce anti-inflammatory substances to reconstruct the homeostasis. Mercury also inhibited arginine generation, which may affect the development of T. tridentatus by disrupting the crucial signaling pathway. The mercury methylation caused enhancement of S-adenosylmethionine to meet the need of methyl donor. The mechanisms described in present study provide new insight into the risk assessment of mercury exposure to T. tridentatus.

Reconstitution of (1→3)-ß-D-glucans measurement system using recombinant Limulus polyphemus Factor G.

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-ß-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and ß from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for ß. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and ß subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and ß subunits for BDG-dependent activation.

Response of moulting genes and gut microbiome to nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus.

Nanoplastics (NPs) and heavy metals are typical marine pollutants, affecting the gut microbiota composition and molting rate of marine organisms. Currently, there is a lack of research on the toxicological effects of combined exposure to horseshoe crabs. In this study, we investigated the effects of NPs and copper on the expression of molt-related genes and gut microbiome in juvenile tri-spine horseshoe crabs Tachypleus tridentatus by exposing them to NPs (100 nm, 104 particles L-1) and/or Cu2+ (10 µgL-1) in seawater for 21 days. Compared with the control group, the relative mRNA expression of ecdysone receptor (EcR), retinoid x receptor (RXR), calmodulin-A-like isoform X1 (CaM X1), and heat shock 70 kDa protein (Hsp70) were significantly increased under the combined stress of NPs and Cu2+. There were no significant differences in the diversity and abundance indices of the gut microbial population of horseshoe crabs between the NPs and/or Cu2+ groups and the control group. According to linear discriminant analysis, Oleobacillus was the most abundant microorganism in the NPs and Cu2+ stress groups. These results indicate that exposure to either NPs stress alone or combined NPs and Cu2+ stress can promote the expression levels of juvenile molting genes. NPs exposure has a greater impact on the gut microbial community structure of juvenile horseshoe crabs compared to Cu2+ exposure. This study is helpful for predicting the growth and development of horseshoe crabs under complex environmental pollution.

A novel cadmium detoxification pathway in Tri-spine horseshoe crab (Tachypleus tridentatus): A 430-million-years-ago organism.

Marine and intertidal heavy metal pollution has been a major concern in recent years. Tachypleus tridentatus has existed on earth for more than 430 million years. It has suffered a sharp decline in population numbers caused by environmental pollution and anthropogenic disturbance for almost 40 years. However, the effects of heavy metal pollution on juvenile T. tridentatus have not been reported. Here we show the mechanism of cadmium (Cd) detoxification in juvenile T. tridentatus using integrated antioxidant indexes and transcriptomic and metabolomic analysis. High Cd2+ concentration caused oxidative stress in juvenile T. tridentatus. The hazards increase with increasing Cd2+ concentration in juvenile T. tridentatus. Transcriptomics and metabolomics analyses concluded that high Cd2+ concentration resulted in the imbalance of glycerophospholipid metabolism in juvenile T. tridentatus to detoxify Cd. Our results offer a rationale for protective measures and further studies of heavy metal stress in T. tridentatus.

Microsatellite Loci of the Atlantic Horseshoe Crab (Limulus polyphemus) Reveal Inter-Localities Genetic Diversity in the Coastal Waters of the Eastern and Northern Yucatan Peninsula.

The American horseshoe crab (Limulus polyphemus) is an economically and ecologically important species, which is currently categorized as endangered in Mexico. L. polyphemus, one of four extant horseshoe crab species that constitute the class Merostomata, is distributed along the Atlantic coastline of the USA from Alabama to Maine and has another population on the coastline of Campeche, Yucatan, and Quintana Roo in the Yucatan Peninsula, Mexico. In the present study, we evaluated the genetic diversity and genetic structure of four separated localities along the coast of the Yucatan peninsula (Champoton, CH; Isla Arena, IA; Rio Lagartos, RL; and Holbox Island, HI), using nine microsatellite-type molecular markers for this species. The aim of this study is to obtain a baseline of the current level of genetic diversity, which would allow the monitoring of important changes over time. Multilocus analyses revealed moderate levels of genetic diversity (He, 0.5230 to 0.6389) and genetic structure within the whole study area (FST 0.025). The population from RL showed limited gene flows, differing significantly from the other sampling sites. The genetic information obtained in this study can support the implementation of management and conservation programs for this species in Mexico.

Coming into clear sight at last: Ancestral and derived events during chelicerate visual system development.

Pioneering molecular work on chelicerate visual system development in the horseshoe crab Limulus polyphemus surprised with the possibility that this process may not depend on the deeply conserved retinal determination function of Pax6 transcription factors. Genomic, transcriptomic, and developmental studies in spiders now reveal that the arthropod Pax6 homologs eyeless and twin of eyeless act as ancestral determinants of the ocular head segment in chelicerates, which clarifies deep gene regulatory and structural homologies and recommends more unified terminologies in the comparison of arthropod visual systems. Following this phylotypic stage, chelicerate visual system development differs fundamentally from other arthropods during the compartmentalization of the ocular segment in that eye and optic neuropil primordia originate independently from each other. Comparative analyses of this phase identified further gene regulatory homologies but also major differences, most notably the possibly compensatory replacement of Pax6 by Pax2 in lateral eye specification. Also see the video abstract here: https://youtu.be/Hdfr3z5kEXU.

Weighted gene co-expression network analysis of embryos and first instar larvae of the horseshoe crab Tachypleus tridentatus uncovers development gene networks.

Horseshoe crabs are marine chelicerates that have existed on Earth for about 450 million years, and they are often used as an experimental model for studying marine invertebrate embryology. In this study, we performed transcriptome gene expression profiling of four continuous embryonic stages (Stages 18-21) and first instar larvae of Tachypleus tridentatus. A mean of 50,742,995 high-quality clean reads was obtained from each library. We then conducted weighted gene co-expression network analysis (WGCNA) for 13,698 genes with fragments per kilobase of exon per million mapped fragments values >5. We identified 17 modules, six of which likely play critical roles in development. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes was performed on the biologically significant modules. We found that several pathways, such as hedgehog signaling pathway, VEGF signaling pathway, dorso-ventral axis formation, may be involved in the embryonic development process of T. tridentatus. We also identified hub genes that were highly connected in the six critical modules. This is the first study to apply WGCNA to horseshoe crabs to identify hub genes that may play critical roles in development, and our results provide new insight into the mechanisms underlying early development in horseshoe crabs.

Comparative transcriptome profiling of horseshoe crab Tachypleus gigas hemocytes in response to lipopolysaccharides.

Horseshoe crabs (HSCs) are living fossil species of marine arthropods with a long evolutionary history spanning approximately 500 million years. Their survival is helped by their innate immune system that comprises cellular and humoral immune components to protect them against invading pathogens. To help understand the genetic mechanisms involved, the present study utilised the Illumina HiSeq platform to perform transcriptomic analysis of hemocytes from the HSC, Tachypleus gigas, that were challenged with lipopolysaccharides (LPS). The high-throughput sequencing resulted in 352,077,208 and 386,749,136 raw reads corresponding to 282,490,910 and 305,709,830 high-quality mappable reads for the control and LPS-treated hemocyte samples, respectively. Based on the log-fold change of > 0.3 or < -0.3, 1338 genes were significantly upregulated and 215 genes were significantly downregulated following LPS stimulation. The differentially expressed genes (DEGs) were further identified to be associated with multiple pathways such as those related to immune defence, stress response, cytoskeleton function and signal transduction. This study provides insights into the underlying molecular and regulatory mechanisms in hemocytes exposed to LPS, which has relevance for the study of the immune response of HSCs to infection.

Horseshoe crab genomes reveal the evolution of genes and microRNAs after three rounds of whole genome duplication.

Whole genome duplication (WGD) has occurred in relatively few sexually reproducing invertebrates. Consequently, the WGD that occurred in the common ancestor of horseshoe crabs ~135 million years ago provides a rare opportunity to decipher the evolutionary consequences of a duplicated invertebrate genome. Here, we present a high-quality genome assembly for the mangrove horseshoe crab Carcinoscorpius rotundicauda (1.7 Gb, N50 = 90.2 Mb, with 89.8% sequences anchored to 16 pseudomolecules, 2n = 32), and a resequenced genome of the tri-spine horseshoe crab Tachypleus tridentatus (1.7 Gb, N50 = 109.7 Mb). Analyses of gene families, microRNAs, and synteny show that horseshoe crabs have undergone three rounds (3R) of WGD. Comparison of C. rotundicauda and T. tridentatus genomes from populations from several geographic locations further elucidates the diverse fates of both coding and noncoding genes. Together, the present study represents a cornerstone for improving our understanding of invertebrate WGD events on the evolutionary fates of genes and microRNAs, at both the individual and population level. We also provide improved genomic resources for horseshoe crabs, of applied value for breeding programs and conservation of this fascinating and unusual invertebrate lineage.

Intestinal Tract Microbe Communities Associated with Horseshoe Crabs from Beibu Gulf, China.

Until now, there has been little research on the intestinal microbial community of horseshoe crabs. To fill this gap, we investigated the microbiome composition of the Chinese horseshoe crab, Tachypleus tridentatus, and the mangrove horseshoe crab, Carcinoscorpius rotundicauda. We sequenced the 16S rRNA gene of intestinal bacterial species and compared the microbial community structure and diversity. Next, we show that the total effective bacterial sequence was 36,865 reads, and the average annotated operational taxonomic unit (OTU) number was 240. Through hierarchical clustering analysis and principal coordinate analysis samples from two horseshoe crab species, we found that the intestinal flora of the same horseshoe crab species was relatively concentrated, while the microbiome of a different horseshoe crab species were significantly separated. Cluster analysis showed that two samples, one from Chinese horseshoe crabs and one from mangrove horseshoe crabs, had similar microbial community structure, while other samples were relatively discrete. The gut microbiota of the mangrove horseshoe crab were dominated by the phyla Tenericutes (42.71%), Firmicutes (24.27%), and Proteobacteria (20.39%), while the top three phyla in the Chinese horseshoe crab intestinal tract were Tenericutes (57.19%), Proteobacteria (22.14%), and Bacteroidetes (7.38%). To intuitively understand the similarity and overlap of the OTU composition of each group, we performed Venn diagram analysis. The two species shared 284 OTUs, accounting for 81.8% of the total. This indicates that although there is high similarity between mangrove and Chinese horseshoe crab in gastrointestinal microbial community structure, there are also some differences, which deserve further discussion.

Seasonal Variation in Reproduction of Horseshoe Crabs (Limulus polyphemus) from the Gulf Coast of Florida.

AbstractThe timing of reproduction is often governed by environmental variables, such as temperature or rainfall. Understanding how environmental variables affect mating dynamics is necessary to predict how systems and populations may adapt to changing environmental conditions and is crucial for management of threatened species. The American horseshoe crab (Limulus polyphemus) ranges from the Yucatan to Maine in distinct populations that differ in their timing of reproduction; while most populations have only one breeding period during the spring, some southern populations have two breeding periods. Here we discuss seasonal patterns of reproduction in a Florida Gulf coast population where horseshoe crabs have two periods of breeding: one in the spring and another in the fall. We used environmental measurements, spawning surveys, mark-recapture, and measurements of adult traits and spawning behavior to compare reproductive parameters between the two spawning seasons over three years. We then evaluated whether environmental conditions affect fall and spring horseshoe crab nesting patterns similarly and whether fall and spring horseshoe crabs should be considered two separate populations. We found significant differences in environmental conditions across seasons and in a wide variety of horseshoe crab traits and nesting parameters. Furthermore, environmental conditions affected nesting behaviors of fall and spring horseshoe crabs differently. However, some individuals spawn during both seasons, suggesting that trait differences may be attributable to environmental effects during development or seasonal plasticity, rather than genetic differences, although further study is necessary. Finally, our results suggest that management practices should be tailored to each population, because environmental conditions may have different effects even on genetically similar groups.

Chromosome-level genome assembly of the coastal horseshoe crab (Tachypleus gigas).

Horseshoe crabs, represented by only four extant species, have existed for around 500 million years. However, their existence is now under threat because of anthropogenic activities. The availability of genomic resources for these species will be valuable in planning appropriate conservation measures. Whole-genome sequences are currently available for three species. In this study, we have generated a chromosome-level genome assembly of the fourth species, the Asian coastal horseshoe crab (Tachypleus gigas; genome size 2.0 Gb). The genome assembly has a scaffold N50 value of 140 Mb with ~97% of the assembly mapped to 14 scaffolds representing 14 chromosomes of T. gigas. In addition, we have generated the complete mitochondrial genome sequence and deep-coverage transcriptome assemblies for four tissues. A total of 26,159 protein-coding genes were predicted in the genome. The T. gigas genome contains five Hox clusters similar to the mangrove horseshoe crab (Carcinoscorpius rotundicauda), suggesting that the common ancestor of horseshoe crabs already possessed five Hox clusters. Phylogenomic and divergence time analysis suggested that the American and Asian horseshoe crab lineages shared a common ancestor around the Silurian period (~436 Ma). Comparison of the T. gigas genome with those of other horseshoe crab species with chromosome-level assemblies provided insights into the chromosomal rearrangement events that occurred during the emergence of these species. The genomic resources of T. gigas will be useful for understanding their genetic diversity and population structure and would help in designing strategies for managing and conserving their stocks across Asia.

Chromosome-level assembly of the horseshoe crab genome provides insights into its genome evolution.

The evolutionary history of horseshoe crabs, spanning approximately 500 million years, is characterized by remarkable morphological stasis and a low species diversity with only four extant species. Here we report a chromosome-level genome assembly for the mangrove horseshoe crab (Carcinoscorpius rotundicauda) using PacBio reads and Hi-C data. The assembly spans 1.67 Gb with contig N50 of 7.8 Mb and 98% of the genome assigned to 16 chromosomes. The genome contains five Hox clusters with 34 Hox genes, the highest number reported in any invertebrate. Detailed analysis of the genome provides evidence that suggests three rounds of whole-genome duplication (WGD), raising questions about the relationship between WGD and species radiation. Several gene families, particularly those involved in innate immunity, have undergone extensive tandem duplication. These expanded gene families may be important components of the innate immune system of horseshoe crabs, whose amebocyte lysate is a sensitive agent for detecting endotoxin contamination.

The draft genome of horseshoe crab Tachypleus tridentatus reveals its evolutionary scenario and well-developed innate immunity.

BACKGROUND: Horseshoe crabs are ancient marine arthropods with a long evolutionary history extending back approximately 450 million years, which may benefit from their innate immune systems. However, the genetic mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear. RESULTS: Here, we describe the 2.06 Gbp genome assembly of Tachypleus tridentatus with 24,222 predicted protein-coding genes. Comparative genomics shows that T. tridentatus and the Atlantic horseshoe crab Limulus polyphemus have the most orthologues shared among two species, including genes involved in the immune-related JAK-STAT signalling pathway. Divergence time dating results show that the last common ancestor of Asian horseshoe crabs (including T. tridentatus and C. rotundicauda) and L. polyphemus appeared approximately 130 Mya (121-141), and the split of the two Asian horseshoe crabs was dated to approximately 63 Mya (57-69). Hox gene analysis suggests two clusters in both horseshoe crab assemblies. Surprisingly, selective analysis of immune-related gene families revealed the high expansion of conserved pattern recognition receptors. Genes involved in the IMD and JAK-STAT signal transduction pathways also exhibited a certain degree of expansion in both genomes. Intact coagulation cascade-related genes were present in the T. tridentatus genome with a higher number of coagulation factor genes. Moreover, most reported antibacterial peptides have been identified in T. tridentatus with their potentially effective antimicrobial sites. CONCLUSIONS: The draft genome of T. tridentatus would provide important evidence for further clarifying the taxonomy and evolutionary relationship of Chelicerata. The expansion of conserved immune signalling pathway genes, coagulation factors and intact antimicrobial peptides in T. tridentatus constitutes its robust and effective innate immunity for self-defence in marine environments with an enormous number of invading pathogens and may affect the quality of the adaptive properties with regard to complicated marine environments.

Single-molecule real-time (SMRT) sequencing facilitates Tachypleus tridentatus genome annotation.

Tachypleus tridentatus is a keystone species in marine ecosystems. Its hemolymph also provides the limulus amebocyte lysate (LAL) for detection of bacterial endotoxin in human medical service. Here we combined SMRT sequencing and Illumina RNA-seq to characterize the novel isoforms, novel genetic loci, fusion isoforms formation and transcriptome structure and further to unveil the transcriptome complexity of T. tridentatus. We identified 26,705 non-redundancy isoforms form 10,919 genetic loci, including 25,713 novel isoforms, 2403 novel genes and 170 fusion isoforms. In addition, 1578 novel genes and 23,172 novel isoforms were annotated in the NR, Pfam, KOG, COG, eggNOG, Swiss-Prot, KEGG and GO databases. Meanwhile, we have obtained 4671 gene family clustering based on genetic loci. Furthermore, there are 17,296, 4887, 1054, and 1435 APAs, AS events, lncRNAs, and TFs were identified in the T. tridentatus long-read transcriptome and the target genes of 1054 lncRNA sequences were also predicted. Overall, our work firstly provided the long-read transcriptome and these data are very necessary to improve the annotation information of T. tridentatus genome and optimize the boundaries of 12,342 original reference annotated genes. Furthermore, these information are a potential resource to study LAL secretion mechanisms in T. tridentatus.