Tachypleus Tridentatus Research Articles

Proteomic Identification of Plasma Components in Tachypleus tridentatus and Their Effects on the Longitudinal Bone Growth Rate in Rats.

Tachypleus tridentatus (T. tridentatus) is a marine animal and traditional Chinese medicine. T. tridentatus plasma is a valuable resource for important medical and health-based functions. In this experiment, in order to evaluate the effect and mechanism of T. tridentatus plasma with respect to the promotion of bone tissue growth in rats, the processes of ultrafiltration and mass spectrometry were first used to separate and identify the components of T. tridentatus plasma. Then, a comparison of the effects of the T. tridentatus plasma samples, which each possessed different molecular weights, regarding the growth of the long bones of rats was conducted. Finally, transcriptomics, proteomics, and bioinformatics were all used to analyze the biological functions and related signaling pathways of the T. tridentatus plasma in order to promote rat bone growth. The results showed that the contents of amino acid residues in peptides are related to the growth promotion that was contained in the 10-30 kDa plasma group. Moreover, the T. tridentatus plasma samples were found to be higher in this respect than those in the whole plasma group. In addition, the 10-30 kDa plasma group could significantly promote bone growth activity in rats. The proteomic analysis showed that the proteins that were differentially expressed in the 10-30 kDa plasma group were mainly enriched in the PI3K-AKT signal pathway. Our study suggested that the T. tridentatus plasma possesses promising potential for the purposes of clinical use, whereby it can serve the role of a growth-promoting agent.

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.

A Comparative Study of Oncolytic Vaccinia Viruses Harboring Different Marine Lectins in Breast Cancer Cells

Our previous studies demonstrated that arming vaccinia viruses with marine lectins enhanced the antitumor efficacy in several cancer cells. This study aims to compare the efficacy of oncolytic vaccinia viruses harboring Tachypleus tridentatus lectin (oncoVV-TTL), Aphrocallistes vastus lectin (oncoVV-AVL), white-spotted charr lectin (oncoVV-WCL), and Asterina pectinifera lectin (oncoVV-APL) in breast cancer cells (BC). These results indicated that oncoVV-AVL elicited the highest anti-tumor effect, followed by oncoVV-APL, while oncoVV-TTL and oncoVV-WCL had lower effects in BC. Further studies showed that apoptosis and replication may work together to enhance the cytotoxicity of oncoVV-lectins in a cell-type dependent manner. TTL/AVL/APL/WCL may mediate multiple pathways, including ERK, JNK, Hippo, and PI3K pathways, to promote oncoVV replication in MDA-MB-231 cells. In contrast, these pathways did not affect oncoVV-TTL/AVL/APL/WCL replication in MCF-7 cells, suggesting that the mechanisms of recombinant viruses in MCF-7 (ER+, PR+) and MDA-MB-231 (TNBC) cells were significantly different. Based on this study, we hypothesized that ER or PR may be responsible for the differences in promoting viral replication and inducing apoptosis between MCF-7 and MDA-MB-231 cells, but the specific mechanism needs to be further explored. In addition, small-molecule drugs targeting key cellular signaling pathways, including MAPK, PI3K/Akt, and Hippo, could be conjunction with oncoVV-AVL to promote breast cancer therapy, and key pathway factors in the JNK and PI3K pathways may be related to the efficacy of oncoVV-APL/TTL/WCL. This study provides a basis for applying oncolytic vaccinia virus in breast carcinoma.

Adverse impacts of high-density microplastics on juvenile growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus

The impacts of high-density microplastics, namely polyamine 6,6 (nylon), polymethyl methacrylate (PMMA) and polyethylene terephthalate (PET), on growth and behaviour of the endangered tri-spine horseshoe crab Tachypleus tridentatus were investigated for 100 days. Negative changes in wet weight and prosomal width of the juveniles were observed in all treatments of microplastics, but significant difference was only detected in prosomal width between control and PMMA. T. tridentatus became significantly less active upon exposure to nylon and PET. The extent of burrowing by T. tridentatus did not significantly differ among the treatments but was overall significantly reduced towards day 100. T. tridentatus exposed to PET significantly showed the lowest survival probability (30 %), compared to the other treatments (70–90 %). In conclusion, high-density microplastics compromised growth and behaviour of juvenile horseshoe crabs. Among the polymers that were tested, PET was considered more harmful and associated with higher mortality.

The current situation of horseshoe crabs in the offshore waters of northern South China Sea with analysis of the potential habitat distribution of juvenile <i>Tachypleus tridentatus</i> in Beibu Gulf

The current situation of horseshoe crabs in the offshore waters of northern South China Sea with analysis of the potential habitat distribution of juvenile <i>Tachypleus tridentatus</i> in Beibu Gulf

Characterization of Trypsin-like Serine Proteases from Tachypleus tridentatus Amebocyte Lysate and its Potential for Endotoxin Detection

Background: Serine Proteases in the Tachypleus Amoebocyte Lysate (TAL) from horseshoe crab, Tachpleus tridentatus have many applications in biomedical industries. The sensitivity of these enzymes is a crucial factor for such usages. Until today, the catalytic behavioural and biochemical stability of these enzymes has not been properly addressed. This report aimed to characterize the physicochemical properties of serine proteases to determine their kinetic reactions and to determine the potential utilization of the enzymes for endotoxin detection. Methods: The purification process of protease from crude TAL was performed using a two-phase filtration unit by affinity and gel columns and the molecular weights of the enzyme fraction from crude, affinity and gel filtration columns were determined. The Azocasein, Aldolase and transaldolase assays were used for the determination of the enzyme Catalysis. The chromogenic assay was carried out to determine the bacterial endotoxin effects using a standard endotoxin from strain Escherichia coli 0113: H10 as a positive control. The absorbance was measured at 405 nm. The enzyme activity was determined using the constructed p-nitroanilide standard curve. Result: A 72 kDa trypsin-like enzyme was purified at fraction-14 (F14) gel filtration and identified as transaldolase. The purified-F14 hydrolysed trypsin substrate, Ná-Benzoyl-L-Arginine-p-nitroanilide at higher activity than chymotrypsin. In conclusion, the purified enzyme showed a great potential for enhancing the TAL sensitivity.

Chemically mediated rheotaxis of endangered tri-spine horseshoe crab: potential dispersing mechanism to vegetated nursery habitats along the coast.

An enhanced understanding of larval ecology is fundamental to improve the management of locally depleted horseshoe crab populations in Asia. Recent studies in the northern Beibu Gulf, China demonstrated that nesting sites of Asian horseshoe crabs are typically close to their nursery beaches with high-density juveniles distributed around mangrove, seagrass and other structured habitats. A laboratory Y-maze chamber was used to test whether the dispersal of early-stage juvenile tri-spine horseshoe crab Tachypleus tridentatus is facilitated by chemical cues to approach suitable nursery habitats. The juvenile orientation to either side of the chamber containing controlled seawater or another with various vegetation cues, as well as their movement time, the largest distance and displacement were recorded. The juveniles preferred to orient toward seagrass Halophila beccarii cues when the concentration reached 0.5 g l-1, but ceased at 2 g l-1. The results can be interpreted as a shelter-seeking process to get closer to the preferred settlement habitats. However, the juveniles exhibited avoidance behaviors in the presence of mangrove Avicennia marina and invasive saltmarsh cordgrass Spartina alterniflora at 2 g l-1. The juveniles also spent less time moving in the presence of the A. marina cue, as well as reduced displacement in water containing the S. alterniflora cue at 1 and 2 g l-1. These results may explain the absence of juvenile T. tridentatus within densely vegetated areas, which have generally higher organic matter and hydrogen sulfide. Early-stage juvenile T. tridentatus are capable of detecting and responding to habitat chemical cues, which can help guide them to high-quality settlement habitats. Preserving and restoring seagrass beds in the intertidal areas should be prioritized when formulating habitat conservation and management initiatives for the declining horseshoe crab populations.

Đa dạng động vật đáy ở sông Chanh, tỉnh Quảng Ninh

The macrobenthos fauna in the Chanh River, Quang Ninh province have been studied for two surveyed, from September 2020 to May 2021. A total of 99 macrobenthos species belonging to three phyla, five class, 19 orders, 37 families and 58 genera were collected and identified. The most dominant phyla of macrobenthos were Mollusca (55 species) followed by Arthropoda (40 species) and Annelida (4 species). Two vulnerable (Tachypleus tridentatus, Charybdis feriata) macrobenthos species of Vietnam Red Data Book (2007) were recorded and one endangered (Tachypleus tridentatus) species was also listed in IUCN Red List (2021).

Design, synthesis and antibacterial activity of a class of novel molecular engineering analogues of Tachyplesin I

Tachyplesin I (TP-I) is a marine-derived antimicrobial peptide isolated from the hemocyte membrane of Japanese horseshoe crab ( Tachypleus tridentatus ) with broad antimicrobial spectrum. It consists of 17 amino acid residues and shows a characteristic structure with three tandem repeats of a tetrapeptide sequence. The three tandem repeats of a tetrapeptide sequence are the key components of the amphipathic β-hairpin structure of TP-I and we proposed that these fragments were critical for the antimicrobial activity, and designed a class of novel molecular engineering analogs of TP-I via re-combination of the three tetrapeptide fragments with different numbers of disulfide bonds in a search of therapeutically valuable lead compound. The synthesis of cyclic peptides with 1/2 disulfide bond(s) are challenging and we successfully prepared most of them using the orthogonal Cys protection strategy. The antimicrobial assay result revealed that the molecular engineering analogs of TP-I via fragment re-combination with 2 disulfide bonds maintained the antimicrobial potency across the range of microbes and confirmed the importance of tetrapeptide fragments and disulfide bonds. Of particular interest, analogue TP-I-R2 exhibited good antimicrobial activity against most of the tested microbes with MIC ranging from 4 to 128 μg/mL. Moreover, TP-I-R1, TP-I-R1b and TP-I-R3 particularly exhibited higher potency of more than 16/16/8-fold against Grampositive Bacillus pumilus strains than TP-I, TP-I-R2 and TP-I-R4 showed comparable potency against Gramnegtive Salmonella typhimurium strains with TP-I. These compounds may be considered as promising lead for further development toward new treatments for infections caused by multiple bacterial pathogens. • A class of novel molecular engineering analogues of Tachyplesin I (TP-I) were designed and synthesized. • TP-I-R2 exhibited broader antimicrobial spectrum and good antimicrobial potency. • TP-I-R2 could be considered as promising lead for the dicovery of novel antibiotics.

Identification and Morphology of the Two Horseshoe Crab Species, Tachypleus tridentatus (Leah, 1819) and Carcinoscorpius rotundicauda (Latreille, 1802) (Merostomata: Limulidae) from Honda Bay, Palawan, Philippines

There were reports of the existence of horseshoe crabs in Palawan, Philippines, but there is a need to identify and confirm these species. A total of 128 adult horseshoe crabs were collected from Honda Bay, Palawan, Philippines. These were identified as Tachypleus tridentatus (68 males, 40 females) and Carcinoscorpius rotundicauda (2 males, 18 females). Morphology, morphometric measurements, and ratios of male and female of each species were described. Morphological characteristics in differentiating a male from a female crab involve examination of the appearance of the genitalia. Morphological characteristics in identifying the species include the presence or absence of notches in the frontal margin, arching in the frontal view, appearance of the chelae of the 2nd and 3rd prosomal appendages, number of lateral spines and the immovable spines in the opisthosoma, the presence of spinnerets on the surface of the telson, and shape of the telson and its cavity in cross-section. This study confirmed the identification of the two species found in Palawan, Philippines, and the morphological characteristics of the male and female of each species. In addition, a comparison was made of the size of the horseshoe crab species in Palawan with those found in other locations in the Asia-Pacific region.

Analysis of the topological motifs of the cellular structure of the tri-spine horseshoe crab (Tachypleus tridentatus) and its associated mechanical properties

Topological motifs in pore architecture can profoundly influence the structural properties of that architecture, such as its mass, porosity, modulus, strength, and surface permeability. Taking the irregular cellular structure of the tri-spine horseshoe crab as a research model, we present a new approach to the quantitative description and analysis of structure-property-function relationships. We employ a robust skeletonization method to construct a curve-skeleton that relies on high-resolution 3D tomographic data. The topological motifs and mechanical properties of the long-range cellular structure were investigated using the Grasshopper plugin and uniaxial compression test to identify the variation gradient. Finite element analysis was conducted for the sub-volumes to obtain the variation in effective modulus along the three principal directions. The results show that the branch length and node distribution density varied from the tip to the base of the sharp corner. These node types formed a low-connectivity network, in which the node types 3-N and 4-N tended to follow the motifs of ideal planar triangle and tetrahedral configurations, respectively, with the highest proportion of inter-branch angles in the angle ranges of 115–120° and 105–110°. In addition, mapping the mechanical gradients to topological properties indicated that narrower profiles with a given branch length gradient, preferred branch orientation, and network connectedness degree are the main factors that affect the mechanical properties. These factors suggest significant potential for designing a controllable, irregularly cellular structure in terms of both morphology and function.

Combined effects of nanoplastics and heavy metal on antioxidant parameters of juvenile tri-spine horseshoe crabs

Juvenile tri-spine horseshoe crabs (Tachypleus tridentatus) were exposed to determine the effects of single and combined stresses of polystyrene nanoplastics (nano-PS) and heavy metal (Cu2+) on antioxidant enzyme parameters. The juveniles were exposed to a 21-day 100-nm polystyrene concentration (104 particles l-1) and a concentration of Cu2+ (10 µg l-1) followed by a recovery period of 7 days. The in vivo antioxidant activity for whole horseshoe crab was analyzed. The results revealed that all antioxidant parameters, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and lipid peroxidation (LPO), showed both increased and decreased levels in different experimental groups of horseshoe crabs having different experimental conditions compared to the control group at three time points, i.e., on days 7, 14, and 21. Similarly during the recovery period, SOD, CAT, and MDA showed decreased levels in all experimental groups, while GSH and LPO showed increased levels in all experimental groups of horseshoe crabs under the influence of different experimental conditions of nanoplastics and heavy metals compared to the control group on day 28. These results showed that the exposure of nano-PS and Cu2+ had precise effects on juvenile horseshoe crabs. Integrated biomarker responses showed that nano-PS and Cu2+ had adverse effects on juvenile horseshoe crabs. By principal component analysis, the potentially toxic effects of nano-PS and Cu2+ on horseshoe crabs were obtained.

Assessment of spawning events of the tri-spine horseshoe crab, <i>Tachypleus tridentatus</i>, using environmental DNA in the Sone tidal flat, Japan

Assessment of spawning events of the tri-spine horseshoe crab, <i>Tachypleus tridentatus</i>, using environmental DNA in the Sone tidal flat, Japan

Impact of Different Diets on Adult Tri-Spine Horseshoe Crab, Tachypleus tridentatus.

Effective culture and management of adult tri-spine horseshoe crab, Tachypleus tridentatus can ensure that stock enhancement programs and aquaculture systems are maintained. To explore suitable feed for animals during the breeding season, Pacific oyster (Ostrea gigas) (oyster group; OG) and frozen sharpbelly fish (Hemiculter leucisculus) (frozen fish group; FG) were selected to feed 20 T. tridentatus male and female pairs, respectively. At the end of the experiment, intestinal samples were obtained to measure digestive enzymes activities. The intestinal flora were determined by 16S rDNA sequencing. No eggs were observed in the FG and one T. tridentatus adult died. No animals died in the OG, and 9.7 × 104 eggs were obtained. These results show that oysters are more suitable for the development and reproduction of adult T. tridentatus than frozen fish. Additionally, the digestive enzyme activity analysis revealed that animals in the OG exhibited higher protein digestibility than those in the FG, but no significant differences in lipid and carbohydrate uptake were observed between the groups. Furthermore, the intestinal flora analysis showed that operational taxonomic units (OTUs) and the Chao1 index were significantly higher in the OG than in the FG, but no significant difference was observed in the Shannon or Simpson indices between the groups. Our data indicate that the oyster diet improved the intestinal microbial diversity of T. tridentatus. We hypothesize that nutrients, such as oyster-based taurine, proteins, and highly unsaturated fatty acids, improve protease activity in the T. tridentatus digestive tract, alter the intestinal floral structure, and improve the reproductive performance of T. tridentatus.

Effect of Intertidal Elevation at Tsuyazaki Cove, Fukuoka, Japan on Survival Rate of Horseshoe Crab Tachypleus tridentatus Eggs.

Physical factors affecting the survival of Tachypleus tridentatus eggs were investigated by translocating their eggs between the high intertidal zone and the low intertidal zone of a known spawning site. The mean egg survival rates per day were highest in the mid intertidal zone (45.1%±25.4%) and the lowest in the low intertidal zone (13.3%±27.6%). Differences in the elevation, air exposure time, and water content of the spawning ground were significant factors determining the egg survival rates. Excessive or insufficient air exposure time resulted in inadequate water content at higher and lower intertidal zones and could reduce egg survival. On the other hand, moderate saturation and dehydration were repeated with each tidal movement in the mid intertidal zone. This dynamic is considered as one of the crucial factors for the survival of eggs and is considered optimal for spawning. Therefore, the protection of the mid intertidal zone is imperative for maximizing the egg survival rate in Tsuyazaki Cove where almost all suitable nesting sites have disappeared due to coastal development. By protecting these optimal sites for spawning and recovering other optimal sites on suitable beaches, a positive contribution can be made to future management and conservation. The study also suggests that translocating eggs from marginal to optimal spawning sites might be a recovery strategy for this globally endangered species.

Identification of a Novel Antimicrobial Peptide From the Ancient Marine Arthropod Chinese Horseshoe Crab, Tachypleus tridentatus.

Humoral immunity is the first line of defense in the invertebrate immune system, and antimicrobial peptides play an important role in this biological process. A novel antimicrobial peptide, termed Tatritin, was identified and characterized in hemolymph of Chinese horseshoe crab, Tachypleus tridentatus, infected with Gram-negative bacteria via transcriptome analysis. Tatritin was significantly induced by bacterial infection in hemolymph and gill. The preprotein of Tatritin consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. The putative mature peptide was 5.6 kDa with a theoretical isoelectric point (pI) of 9.99 and showed a α-helix structure in the N-terminal and an anti-parallel β-sheet structure in the cysteine-stabilized C-terminal region. The chemically synthesized peptide of Tatritin exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Furthermore, Tatritin may recognize and inhibit pathogenic microorganisms by directly binding to LPS, DNA, and chitin. In addition, administration of Tatritin reduced the mortality of zebrafish after bacterial infection. Due to its broad-spectrum antimicrobial activity in vivo and in vitro and the sensitivity to drug-resistant bacterial strains, Tatritin peptide can be used as a new type of drug for infection treatment or as an immune enhancer in animals.

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.

Effects of Ocean Acidification on Molting, Oxidative Stress, and Gut Microbiota in Juvenile Horseshoe Crab Tachypleus tridentatus.

Anthropogenic elevation of atmospheric carbon dioxide (CO2) drives global-scale ocean acidification (OA), which has aroused widespread concern for marine ecosystem health. The tri-spine horseshoe crab (HSC) Tachypleus tridentatus has been facing the threat of population depletion for decades, and the effects of OA on the physiology and microbiology of its early life stage are unclear. In this study, the 1st instar HSC larvae were exposed to acidified seawater (pH 7.3, pH 8.1 as control) for 28 days to determine the effects of OA on their growth, molting, oxidative stress, and gut microbiota. Results showed that there were no significant differences in growth index and molting rate between OA group and control group, but the chitinase activity, β-NAGase activity, and ecdysone content in OA group were significantly lower than those of the control group. Compared to the control group, reactive oxygen species (ROS) and malondialdehyde (MDA) contents in OA group were significantly increased at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatase (AKP) activities increased first and then decreased, glutathione peroxidase (GPX) decreased first and then increased, and GST activity changed little during the experiment. According to the result of 16S rRNA sequencing of gut microbiota, microbial-mediated functions predicted by PICRUSt showed that “Hematopoietic cell lineage,” “Endocytosis,” “Staphylococcus aureus infection,” and “Shigellosis” pathways significantly increased in OA group. The above results indicate that OA had no significant effect on growth index and molting rate but interfered with the activity of chitinolytic enzymes and ecdysone expression of juvenile horseshoe crabs, and caused oxidative stress. In addition, OA had adverse effects on the immune defense function and intestinal health. The present study reveals the potential threat of OA to T. tridentatus population and lays a foundation for the further study of the physiological adaptation mechanism of juvenile horseshoe crabs to environmental change.

Structural characterization and regulation of the mechanical properties of the carapace cuticle in tri-spine horseshoe crab (Tachypleus tridentatus)

Horseshoe crab (order Xiphosura) has a large and thick carapace that has evolved as a protective tool to defend against predators and resist impacts from surf-zone turbulence. The naturally occurring spatial variation in the mechanical properties of the carapace cuticle need to be investigated to understand their regulatory mechanism and the underlying design strategies. In this work, we used a combination of high-resolution optical microscopy, scanning electron microscopy, (SEM) and energy-dispersive X-ray spectroscopy (EDS) to evaluate the multiscale microstructure and elemental composition of the cuticle of tri-spine horseshoe crab (Tachypleus tridentatus). The moduli, ultimate strengths, and failure strains of the three individual layers and the entire cuticle were systematically characterized in both the dry and hydrated states. The failure behaviors and energy absorption of the cuticle involved stress stiffening, toughness mechanism and environmental adaptation were analyzed qualitatively and quantitatively and then correlated with the morphological features in different cuticle regions. The mechanical properties are primarily influenced by the endocuticle thickness ratio; a higher thickness ratio corresponds to more stacking of the vertical lamellae, leading to a lower modulus, weaker strength, and greater elongation of the endocuticle. Radial energy is absorbed primarily by the endocuticle, with the energy absorbed in the radial direction being nearly twice that absorbed in the circumferential direction. This is attributed to the larger failure strain and relatively small decrease in the stress plateau in the radial direction. The findings provide a deeper understanding of how nature modulates the cuticle's mechanical properties and inspiration for developing high-performance synthetic composites.

DNA Metabarcoding Revealed Interspecific Dietary Difference and Prey Selectivity in Juvenile Horseshoe Crabs Carcinoscorpius rotundicauda and Tachypleus tridentatus From Hong Kong

Horseshoe crabs provide important ecological services including bioturbation and linking of food web in the shallow waters, but their populations are declining globally, leading to major concerns on conservation of these iconic animals. Baseline information of horseshoe crab ecology, such as their trophic role and food source, is pre-requisite for habitat protection plan and captive restocking program. Trophic ecology of Asian horseshoe crabs is relatively poorly understood and previous studies on their juveniles have suggested that they are selective feeders rather than opportunistic generalists. This study demonstrates a non-invasive approach, using DNA metabarcoding analyses of the nuclear 18S rRNA gene on fecal samples to assess the dietary compositions of Carcinoscorpius rotundicauda and Tachypleus tridentatus juveniles to (1) determine their dietary compositions and trophic roles in their ecosystem, (2) determine any prey selectivity, and (3) distinguish the interspecific dietary differences with potential implications on the habitat requirement and ecological partitioning between these two horseshoe crab species. The results based on relative read abundance (RRA) suggested that oligochaetes were the major prey items for both C. rotundicauda (41.6%) and T. tridentatus (32.4%). Bivalves and crustaceans were second major prey groups for C. rotundicauda (8.6 and 8.4%, respectively). Surprisingly, anthozoans contributed a considerable portion of T. tridentatus’s diet (22.8%), which has never been reported. Furthermore, the major prey groups identified in the fecal samples were not the dominant benthic organisms in the studied area as revealed by environmental DNA (eDNA) analyses on the sediment samples, implying that both species are selective feeders rather than dietary generalists. Significant differences observed in the dietary compositions of the two species might be partially due to the difference in habitat preference between the two species. This study provides new insights into the trophic ecology of the two Asian horseshoe crab species in the estuarine habitat and establishes a new framework for future detailed molecular dietary analyses on all developmental stages of horseshoe crabs around the world, which will allow us to evaluate the food sources needed for the survival of horseshoe crabs and facilitate future conservation strategies without killing the animals.