Asian Horseshoe Crabs Research Articles

Potential role of the antimicrobial peptide Tachyplesin III in regulating nontypeable Haemophilus influenzae-induced inflammation in airway epithelial cells.

The respiratory bacterium nontypeable (non-encapsulated) Haemophilus influenzae (NTHi) is a key pathogen driving exacerbations in chronic obstructive pulmonary disease (COPD), and is associated with an excessive airway inflammation. Increasing issues with tolerance and unwanted side effects of existing pharmaceuticals present an urgent need for new, effective and minimally toxic therapeutic options. This study aimed to investigate the potential role of Tachyplesin III, an antimicrobial peptide derived from the hemolysates of Southeast Asian horseshoe crabs, in regulating NTHi-induced airway inflammation. The results revealed that Tachyplesin III effectively inhibited the production of IL-1β in NTHi-stimulated human lung epithelial cells (A549), without causing cytotoxic effects. Additionally, Tachyplesin III significantly reduced TNF-α, PGE2 and NO production in NTHi-stimulated A549 cells. Moreover, this peptide inhibited the nuclear translocation of the NF-κB p65 subunit in NTHi-stimulated lung epithelial cells. It also reduced transcriptional activation of NF-κB target genes, as shown by lower mRNA levels of IL-1β, TNF-α, COX-2 and iNOS, which correlated with corresponding decreases in their protein expression. Tachyplesin III peptide also inhibited pro-IL-1β and NLRP3 protein expression and prevented NTHi-induced caspase-1 cleavage and IL-1β maturation. Together, our findings demonstrate that Tachyplesin III effectively reduced NTHi-mediated inflammation via the NF-κB/NLRP3 inflammasome signaling pathway, highlighting its important anti-inflammatory activity. Complementing these findings, in silico analysis revealed key pharmacokinetic and toxicological attributes, establishing a foundational understanding of Tachyplesin III as a promising therapeutic agent for managing NTHi-associated inflammation.

Population structure and feeding habits of Carcinoscorpius rotundicauda in the bay environment, Southern Gulf of Thailand

This study presents the first comprehensive assessment of the population structure and feeding habits of Carcinoscorpius rotundicauda in Thailand. Sample collection was conducted using crab gill nets at seven sampling sites characterized by distinct ecosystem attributes in Pattani Bay between February 2019 and March 2020. Our findings indicate the presence of a single Asian horseshoe crab (HSC) species within the bay, with a sex ratio of approximately 1:1 (48.1 % males, 51.9 % females) that did not deviate significantly from parity. Habitat variation within the bay did not influence HSC abundance (P > 0.05) whereas monthly abundance fluctuations were highly significant (P < 0.001), peaking in March and reaching the lowest in September. Positive correlations were observed between HSC abundance and environmental factors including dissolved oxygen, temperature, water pH, water depth, and chlorophyll c concentrations. Linear regression analysis revealed a positive correlation between morphometric characteristics and body weight. However, multiple linear regression analysis indicated a negative relationship between body weight and telson length (TEL) and opisthosoma width (OW) of males, as well as TEL of females. The size at which 50 % of the HSC population reached maturity (L50) was estimated to be 12.2 cm for males and 13.5 cm for females based on prosomal width (PW). The average PW measurements of HSC at different egg stages (1–4) from developing to mature females was 14.4 cm, with corresponding body weights ranging from 224 to 243 g. Females at egg of stages 3 and 4 were found throughout the year, indicating multiple spawning events, particularly prevalent in January-April, and predominantly abundant at stations 1, 2, 5 and 6. HSC primarily consumed bivalves (22.1±21.2 %), followed by fishes (5.9±13.0 %) and crustaceans (2.9±9.0 %) by volumetric contribution (%V). This comprehensive study provides novel insights into the population dynamics and feeding ecology of HSC within Thailand. The findings establish a foundational framework for future research endeavors and offer critical data to inform the management and conservation of their habitats.

Genetic differentiation and heavy metal pollution influence on horseshoe crab Tachypleus gigas populations in Peninsular Malaysia: A comprehensive allozyme analysis

This study aimed study the genetic diversity and differentiation of Asian horseshoe crab Tachypleus gigas (Müller, 1785) populations across three distinct locations in Peninsular Malaysia: Kampung Pasir Puteh (KPPuteh), Kuala Sungai Ayam (KSAyam), and Kampung Pulau Sayak (KPSayak). A total of 28 individuals were collected, and their genetic structure was analyzed using starch gel electrophoresis. The study employed multiple buffer systems optimized for separating 20 different enzymes, each contributing to understanding the genetic variability within and between populations. The findings of this study, which revealed significant genetic differentiation, particularly between populations in polluted and less impacted environments, as reflected by high FST values and distinct clustering in the UPGMA dendrogram, are of paramount importance. The results indicate that environmental factors, especially metal-polluted sites at KPPuteh and KSAyam, have played a critical role in shaping the genetic structure of these populations. Populations from the two polluted sites, exhibited higher genetic diversity and greater deviations from Hardy–Weinberg equilibrium, suggesting localized selection pressures. Conversely, from a less polluted site, the unpolluted site at KPSayak population showed lower genetic variability and was genetically distinct from the others. These findings underscore the importance of considering environmental influences in conservation strategies for T. gigas, highlighting the need for site-specific management to preserve genetic diversity and ensure long-term survival of the species.

Modeling potential habitats of threatened horseshoe crabs in Indo-Pacific Asia: Suggestions for conservation management

Identifying critical habitats of threatened marine species is key to area-based management measures, while it can be of great challenge for those species that distribute continentally. Asian horseshoe crabs, including tri-spine horseshoe crab Tachypleus tridentatus, coastal horseshoe crab T. gigas, and mangrove horseshoe crab Carcinoscorpius rotundicauda, are globally threatened marine chelicerates, which are largely distributed in developing countries in Indo-Pacific Asia with limited baseline information. Here, we performed the maximum entropy model (MaxEnt), a widely used correlative species distribution model, to explore potential habitats of the Asian horseshoe crabs. Presence-only data of the adult Asian horseshoe crabs and the seven sea benthic environmental predictors, which included temperature, salinity, chlorophyll a concentration, dissolved oxygen, current velocity, bathymetry, and slope, were input into the MaxEnt. The Cloglog output format was adopted to estimate the presence probability of these three species under species-specific regularization multipliers. Our results showed that all three species have potential habitats in Southeast Asia. The range of potential habitats is the most widespread for T. tridentatus, but the most restricted for T. gigas. Bathymetry, temperature, and chlorophyll a concentration are the main contributing environmental predictors for the projected distribution of T. tridentatus, while for T. gigas and C. rotundicauda, temperature and bathymetry have contributed the most. Temperature of the potential habitats is the lowest for T. tridentatus (25.6 ± 4.4 °C), compared to the more tropically distributed T. gigas and C. rotundicauda (around 29 °C). Core habitats of the estuary-bound C. rotundicauda have shallower bathymetry of 5.8 ± 3.5 m in depth. Overall, Asian horseshoe crab species share similar niches, with the common characteristics of shallow bathymetry (<15 m in depth), high temperature (>25 °C), and relatively higher chlorophyll a concentration in coastal waters. We conclude that the projected habitats serve as a critical reference for the conservation management of threatened Asian horseshoe crabs under extensive baseline gaps. Generalized conservation strategies are suggested, depending on the sufficiency of baseline data, to facilitate the formation of specific decision-making.

COI Gene Analysis of Asian Horseshoe Crab in Banyuasin Estuarine Waters, Sumatra, Indonesia

There is limited study on DNA barcoding of horseshoe crabs in South Sumatra, Indonesia. The present study was the first to record horseshoe crabs' COI DNA barcoding in three localities of Banyasin estuarine waters. The nucleotide composition was in a strong adenine thymine bias (AT = 63.90%) with high haplotypic diversity (Hd = 0.858) relative to low nucleotide diversity (π = 0.0534). A total of 16 haplotypes were recorded, of which two were in Carat Cape, 4 in Makati Jaya, and 12 in the Banyuasin river estuary (BRE), and Hap 1 was a dominant type (62.5%). The population differentiation (FST) value was varied, and only in the comparison of BRE-Carat Cape was significant (FST = 0.637), while the gene flow (Nm) value in Makati Jaya-BRE was high (6.563). The neutrality test, Tajima's D (0.4142), and Fu's Fs values were negative (-0.492), suggesting these populations have experienced a current bottleneck or multiplication. Analysis of molecular variation (AMOVA) suggested that 68% was distributed within populations. The haplotype network, PCoA, and phylogenetic of COI DNA sequences clustered in three groups correspond to Carcinoscorpius rotundicauda, Tachypleus gigas, and Tachypleus tridentatus. This finding is a matter of concern for managing and conserving horseshoe crab species in Banyuasin estuarine waters.

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.

Tetrodotoxin in Asian horseshoe crabs Carcinoscorpius rotundicauda and Tachypleus tridentatus across different life stages from northern Beibu Gulf, China.

Horseshoe crabs (HSCs) are a group of ancient chelicerates with great ecological and biomedical importance. Food poisonings caused by the consumption of Asian HSCs have significant impacts on public health and safety. This study measured tetrodotoxin (TTX) concentrations in two HSC species across various life stages in May 2020 from the northern Beibu Gulf, their most important spawning and nursery habitats in China. The average TTX contents in both Carcinoscorpius rotundicauda and Tachypleus tridentatus ranged 6.2-8.0μg/kg and 3.8-8.4μg/kg, respectively. While sampling location, growth and molt stages have little influence on TTX distribution in both species, significantly higher levels of TTX were detected in hemolymph, but lower in pooled tissues of early-instar juvenile T. tridentatus. These results provide a regional view of TTX occurrence and distribution in HSCs during their spawning season, which are critical for future studies to enhance understanding of TTX dynamics and formation in HSCs.

Spatiotemporal Distribution of Asian Horseshoe Crab Eggs Are Highly Intermingled with Anthropogenic Structures in Northern Beibu Gulf, China.

Identification, protection and restoration of spawning habitats are vital for protecting the depleted species. Asian horseshoe crabs are ecologically important macroinvertebrates in coastal and estuarine ecosystems. However, their spawning habitat studies were limited to several reports in tropical regions, possibly due to the lack of modified survey methods, particularly in habitats with a lower density of spawning adults, and/or intermingled with anthropogenic structures. In this study, the year-round egg distribution and spawning habitat baselines of Carcinoscorpius rotundicauda and Tachypleus tridentatus were determined in the northern Beibu Gulf, China. Our findings demonstrated that the peak spawning occurred in June–July and ceased in November–January when the average water temperature dropped below 20 °C. Egg aggregations were found < 10 cm beneath the sediment surface with regular tidal inundation, regardless of seasonal changes, in the vicinity of natural and artificial structures with elevated, mildly sloping substratum within the high tide zones. The nests were characterized by medium-sized sediment grains (0.5–0.9 mm), high temperatures (31–34°C), low water contents (0.8%–0.9%), and total organic carbon contents (0.5%–0.7%), which might maximize the hatching success. The identified nesting beaches were close to nursery habitats for juveniles, and tidal creeks were present as the possible corridor connecting these two important habitats through the dominant mangrove forests. The findings provide valuable insights in the scope of spawning behavior and nest-site selection of Asian horseshoe crabs under a mixture of natural and artificial structures, which could benefit future management efforts for the exploited spawning populations.

Asian Horseshoe Crab Bycatch in Intertidal Zones of the Northern Beibu Gulf: Suggestions for Conservation Management

Asian Horseshoe Crab Bycatch in Intertidal Zones of the Northern Beibu Gulf: Suggestions for Conservation Management

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.

The different fates of two Asian horseshoe crab species with different dispersal abilities.

Impending anthropogenic climate change will severely impact coastal organisms at unprecedented speed. Knowledge on organisms’ evolutionary responses to past sea‐level fluctuations and estimation of their evolutionary potential is therefore indispensable in efforts to mitigate the effects of future climate change. We sampled tens of thousands of genomic markers of ~300 individuals in two of the four extant horseshoe crab species across the complex archipelagic Singapore Straits. Carcinoscorpius rotundicauda Latreille, a less mobile mangrove species, has finer population structure and lower genetic diversity compared with the dispersive deep‐sea Tachypleus gigas Müller. Even though the source populations of both species during the last glacial maximum exhibited comparable effective population sizes, the less dispersive C. rotundicauda seems to lose genetic diversity much more quickly because of population fragmentation. Contra previous studies’ results, we predict that the more commonly sighted C. rotundicauda faces a more uncertain conservation plight, with a continuing loss in evolutionary potential and higher vulnerability to future climate change. Our study provides important genomic baseline data for the redirection of conservation measures in the face of climate change and can be used as a blueprint for assessment and mitigation of the adverse effects of impending sea‐level rise in other systems.

In vivo immunostimulatory effect of the amoebocyte lysate and plasma of Asian horseshoe crab, Tachypleus gigas in a piscine model.

Antimicrobial proteins/peptides are becoming a new generation of immunostimulants for prevention and disease control in human and animals, including aquatic animals. As the haemolymph of horseshoe crabs (Tachypleus) contains broad ranges of bioactive compounds, we have explored the in vivo immunostimulating potential of amoebocyte lysate and plasma using a fish model. Indian major carp, Labeo rohita, yearlings were injected intraperitoneally with two doses of lysate and plasma at 50 and 100µg protein per fish. No abnormalities and/or mortalities were recorded in any group. L. rohita injected with 50µg lysate and 100µg plasma protein showed significant enhancement (P < 0.01) of various haematological and immunological parameters. There was a significant rise in the total protein and globulin content, myeloperoxidase and respiratory burst activity following injection with 50µg lysate and 100µg plasma protein. The agglutinating and haemagglutinating activities were increased albeit not significantly (P > 0.01) in any groups. On the contrary, a significantly high hemolysin titre was recorded in fish that received 100µg plasma protein. Following challenge with Aeromonas hydrophila, both lysate and plasma protein(s) cross protected the fish after 30days. The highest survival (50%) was recorded in group injected with 50µg lysate protein, followed by 45% in both 100µg lysate and plasma protein injected groups.

Characterization of Purified Tachystatin-A2 Isolated from Amoebocytes of Asian Horseshoe Crab, Tachypleus gigas as Potential Antibacterial Peptide

Characterization of Purified Tachystatin-A2 Isolated from Amoebocytes of Asian Horseshoe Crab, Tachypleus gigas as Potential Antibacterial Peptide

Short communication – The first inventory of the nest placement of horseshoe crab (Tachypleus gigas) in Indonesia

Previous studies have reported that extant horseshoe crabs, including Atlantic and Asian horseshoe crab, spawned in intertidal zone during high tide season. Limited information about spawning nest of the horseshoe crab especially Tachypleus gigas in Indonesia has not been known yet. Moreover, according to the IUCN, the conservation status of T. gigas is still data deficient. During the fieldwork, we found the location that indicated as the spawning ground or nest placement of T. gigas. This is the first time when the spawning area of horseshoe crab is found in Subang, Indonesia. There were three clutches of eggs and the total number was 375-534 eggs in varying depth (5-20 cm) below the sand. The second observation in the same location on January 16, 2020, found a pair of T. gigas laying their eggs and the number was 244 eggs in varying depth (5-15 cm). The characteristics of nest placement of T. gigas that found in Subang was located on the small island in the middle of the sea. The island is an uninhabited island that has mangrove area and sandy substrate. Thus, based on what was found Burung island indicated as the spawning ground of coastal horseshoe crab (T. gigas).

2-DIMENSIONAL GEL ELECTROPHORESIS PROFILES OF HEMOLYMPH OF HORSESHOE CRABS TACHYPLEUS GIGAS AND CARCINOSCORPRIUS ROTUNDICAUDA

Horseshoe crabs are members of the Limulidae family and are considered 'living fossils' as these have undergone very little change from their fossilized ancestors in the last 450 million years (Kin and Blazejowski 2014). Presently, four species of horseshoe crabs are found in the world. The Atlantic horseshoe crab Limulus polyphemus and the Asian horseshoe crabs Tachypleus gigas , Tachypleus tridentatus , and Carcinoscorpius rotundicauda out of which Tachypleus gigas and Carcinoscorprius rotundicauda are found in the coast of Balasore, Odisha (Pati et al., 2015; Pati and Dash 2016). While the biology of the Atlantic Horseshoe Limulus polyphemus has been extensively studied and its blood (hemolymph) has found a number of medical applications, very less studies have been carried out on its Asian counterparts. In this context, a preliminary study has been carried out to generate the one and two-dimensional gel electrophoresis profiles of the hemolymph proteins of Tachypleus gigas and Carcinoscorprius rotundicauda .

Nursery habitat of three species of juvenile Asian horseshoe crabs in Teritip Beach, East Kalimantan, Indonesia: Characterization and implication

Documenting juveniles’ presence is a crucial criterion for ensuring that a protected area can support robust populations of horseshoe crabs. Yet, there are no studies on the juvenile of Asian horseshoe crab in Indonesia. As the first report on the juvenile of Asian horseshoe crabs in Indonesia, this study aims to analyze the population density of the juvenile Asian horseshoe crab and the characteristics of nursery habitats in Teritip Beach, East Kalimantan, Indonesia. The discovery of the juvenile of three species (Carcinoscorpious rotundicauda, Tachypleus tridentatus, and T. gigas) was consistent and supporting the existence of adult individuals. Their population density was mainly dominated by C. rotundicauda (1.56–12.50 Ind./100 m2), followed by T. gigas (1.56–3.13 Ind./100 m2) and T. tridentatus (1.56–3.13 Ind./100 m2). The distribution pattern of C. rotundicauda showed that the juveniles were near the mangrove habitat during August–September and relatively seaward during October. Meanwhile, T. gigas and T. tridentatus exhibited a shift in the distribution pattern from September to October. The above results were supported by the most influential habitat parameter, particularly for silt content from the sediment. Based on the population density and habitat characteristics, 0.4 km2 of the intertidal zone was identified as a nursery/spawning ground. These findings are essential drivers for future marine protected areas (MPAs) proposals for horseshoe crabs.

Hemolymph Proteomics and Gut Microbiota of Horseshoe Crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda

Horseshoe crabs are a group of marine chelicerates that contain only four extant species, some of which are endangered. Their hemolymph has been widely used in medical applications for endotoxin detection. Nevertheless, there is limited information on the profiles of their hemolymph proteins and their gut microbial diversity. In this study, we performed the first detailed investigation of the hemolymph proteomics and gut microbiota of two Asian horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda. Among the identified proteins being cataloged in the juvenile and adult hemolymph, unexpectedly, sesquiterpenoid signaling pathway proteins including Heat shock protein 83 (HSP83), Chd64, and a juvenile hormone binding protein (JHBP) were revealed. This provides evidence for the presence of functional sesquiterpenoid hormonal systems in these marine chelicerates. consumption of certain horseshoe crab species often leads to tetrodotoxin poisoning and the horseshoe crab is thought to possess a tetrodotoxin resistance mechanism. As such, sodium channels were analyzed and found to have critical amino acid residues that are similar to the toxin resistant pufferfish sodium channels. The source of the toxin is unknown so we investigated the gut microbiota, and found that Clostridium and Vibrio were the most dominant bacteria in T. tridentatus and C. rotundicauda, respectively. Together, this study provides a framework for further understanding of sesquiterpenoids and gut microbiota of these marine chelicerates.

Future of Asian horseshoe crab conservation under explicit baseline gaps: A global perspective

Horseshoe crabs are currently threatened by escalating human stresses along the Indo-Pacific coastline. Conservation of their remaining populations, however, is hindered by limited baselines throughout their ranges. We performed a questionnaire survey of conservation experts from diverse geographic regions to identify baselines, prominent threats, impediments and alternative strategies that prioritize Asian horseshoe crab conservation. Despite long-term monitoring across habitat types is lacking, local population declines were widely perceived. Ongoing residential/commercial development along the coast was regarded as the most serious threat. A combination of top-down institutional management and bottom-up stakeholder participation was cited as the most promising strategy. Population and habitat baseline collections should be prioritized in future research to inform conservation planning. In this paper, we summarize the survey findings into the BTPAE (Baseline, Threat, Purpose, Action and Evaluation) framework, which constitutes to our best hope for the future of Asian horseshoe crab conservation.

Conservation of Asian horseshoe crabs on spotlight.

Conservation of Asian horseshoe crabs on spotlight.

Ontogenetic resource use and trophic dynamics of endangered juvenile Tachypleus tridentatus among diversified nursery habitats in the northern Beibu Gulf, China.

Horseshoe crabs, the most well-known example of "living fossils", are iconic and ecologically important macroinvertebrates in coastal and estuarine ecosystems. Their blood is a crucial resource for manufacturing Limulus or Tachypleus amebocyte lysate to detect bacterial endotoxins or fungal contamination in drug and medical devices. An enhanced understanding of their ecological roles and trophic interactions in the food webs is fundamental to facilitate resource management for the declining populations in Asia. Foraging information of the Asian species, however, is mainly derived from preliminary, scattered reports from a limited number of study locations. In this study, resource utilization, trophic niche dynamics, and trophic interaction of the juvenile tri-spine horseshoe crab, Tachypleus tridentatus (instars 1-12, approximately 0.5-8years old) across ontogeny was assessed in diversified nursery habitats along the northern Beibu Gulf, China, using carbon and nitrogen stable isotopes. Our results suggest that: (i) T. tridentatus are ecological generalists given the vast range of carbon isotopic values and trophic niche width estimates exhibited between multiple instar groups; (ii) juvenile T. tridentatus across most habitat types predominantly assimilated energy from a variety of basal production sources in the food web, but primarily depended on sedimentary organic matter and seagrass resource pools; (iii) ontogenetic shifts in juvenile dietary proportions were evident, with decreased reliance on sedimentary organic matter, coupled with increased reliance on benthic macroinvertebrate grazers, detritivores, and omnivores with age; and (iv) nearly all juvenile instars occupied similar trophic positions in the food web with slight shifts in trophic position present with increasing size. Our findings indicate that resource availability and ontogenetic diet shifts strongly influence horseshoe crab trophic dynamics, and age should be accounted when formulating habitat conservation measures based on resource use for Asian horseshoe crabs.