Horseshoe Crab Species Research Articles

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.

Mapping the habitat distribution of horseshoe crabs (Tachypleus gigas) in Pamekasan, Madura Island, Indonesia

Abstract Horseshoe crabs, often known as Belangkas or Mimi, is one of the ancient marine faunas with protected conservation status. Horseshoe crabs are distributed in coastal waters of Indonesia of the islands of Sumatra, Java, Kalimantan, and Sulawesi, which are often caught as bycatch. Horseshoe crabs’s bloods were uniquewhich contain amebocytes as a test material for the presence of endotoxin bacteria. The purpose of this study was to identify the species of horseshoe crabs and to analyze environmental conditions, as well as the distribution of horseshoe crabs in the coastal waters of Pamekasan, Madura Island. This study was conducted in June 2023 by collecting data from crab fishers. ArcGIS was applied to delineate the distribution of horseshoe crab. The identification results showed that there was one species horseshoe crab of the Tachypleus gigas in the waters of Polagan Village, Galis District, Pamekasan Regency. The environmental condition of the waters in Polagan Village, Galis District, and Pamekasan Regency was relatively good. Habitat mapping is one way to protect these animals from the threat of extinction and support conservation efforts.

Haplotype diversity of coastal horseshoe crab (Tachypleus gigas, Muller 1785) based on 16S rRNA gene markers as a basis for conservation

Abstract Tachypleus gigas is one of horseshoe crab species found in Indonesia, but does not have sufficient molecular biology information available. This study aims to predict the populations health of T. gigas from the waters of Langkat, Semarang, and Bintan; through analysis of haplotype diversity based on 16S rRNA gene markers. The observation was conducted from November 2022 until April 2023. Sample’s total DNA was isolated, extracted, amplified and then sequenced to obtain the nucleotide base sequence. The BLASTn results showed that the samples were validated as Tachypleus gigas and the genetic distance between locations showed a close kinship relationship between the three. The population health of the horseshoe crabs from Langkat and Semarang were indicated good with high haplotype diversity values, meanwhile the Bintan indicated poor health with low haplotype diversity values. This study of haplotype diversity provided the starting point to the conservation of horseshoe crab T. gigas

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.

Evolutionary Conservation and Diversification of Five Pax6 Homologs in the Horseshoe Crab Species Cluster

Evolutionary Conservation and Diversification of Five Pax6 Homologs in the Horseshoe Crab Species Cluster

GC–MS and ADME profile analysis of Carcinoscorpius rotundicauda bioactive compounds and their potential as COVID-19 antiviral

BackgroundCarcinoscorpius rotundicauda is a species of horseshoe crab that is rarely studied. This animal is often used as part of the blood as a medical sterilization material. This study aims to identify the content of bioactive compounds and their bioactivity as an antiviral for COVID-19. The stages of this research include extraction, GC–MS analysis, molecular docking analysis, and ADME analysis.ResultsThe results showed that in the ethyl acetate extract of the meat, there were 13 bioactive compounds with dominant compound which is propanoic acid (32.15%). Based on molecular docking, one potential compound was found as an antiviral for COVID-19 ACE2, 3CLpro, and RdRp inhibitor, namely 2-methyl-5-(4′-methyl phenyl)sulfonyl-4-nitroimidazole. The druglikeness and ADME compound profile shows support as an excellent oral drug compound.ConclusionsCarcinoscorpius rotundicauda has potential as an inhibitor of ACE2, RdRp, and 3CLpro receptor as an anti-SARS-CoV-2. Further research, such as in vitro and in vivo, is still needed to develop its potential as a COVID-19 antiviral.

Barriers to the Use of Recombinant Bacterial Endotoxins Test Methods in Parenteral Drug, Vaccine and Device Safety Testing.

The Bacterial Endotoxins Test (BET) is a critical safety test that is used to detect bacterial endotoxins, which are the major contributor to fever-inducing contamination risks known as pyrogens. All parenteral therapies, including every lot of injected drugs, vaccines, medical devices, must be tested for pyrogens to ensure patient safety. Bacterial endotoxins test methods were developed as a highly sensitive detection method for bacterial endotoxins, after the discovery of a clotting cascade in horseshoe crab blood. However, horseshoe crab species are limited to some inshore coastal habitats along the Atlantic coast of the USA and others throughout Asia. Fully functional horseshoe crab clotting factors can be manufactured via recombinant protein production, and several BET methods featuring recombinant horseshoe crab proteins have now been developed for commercial use. Recombinant Bacterial Endotoxins Test (rBET) methods based on the use of recombinant Factor C (rFC) were established in the European Pharmacopoeia - however, these methods have not yet been granted compendial status in the United States Pharmacopoeia (USP). In order to facilitate dialogue between stakeholders, the Physicians Committee for Responsible Medicine hosted two virtual roundtable discussions on the perceived barriers to the use of rBET methods for US FDA requirements. Stakeholders agreed that multiple rFC-based methods have been demonstrated to have suitable analytical performance, as described in ICH Q2 on the Validation of Analytical Procedures and USP <1225> on the Validation of Compendial Procedures. United States Pharmacopoeia compendial inclusion of the rFC-based and other rBET methods was favoured, in order to reduce the additional burdens created by a lack of global harmonisation on BET testing requirements.

Comparison on Total Phenolics and Flavonoids and Antioxidant Activities of Methanol Extract of Horseshoe Crab (Tachypleus gigas) Eggs

The marine environment can be a source of abundant bioactive compounds. One of the horseshoe crab species scattered in Indonesian sea waters is Tachypleus gigas. It was reported that the eggs of T. gigas contained flavonoids, saponins, alkaloids, and steroids. Flavonoids are polyphenol compounds that have the ability as natural antioxidants. In this study, total phenolics, flavonoids, and antioxidant activity tests were carried out on the methanol extract of T. gigas eggs. The total phenolics content used the Folin Ciocalteu method, the total flavonoids used the aluminum chloride colorimetric method, and the antioxidant activity test used the FRAP and DPPH methods. The test results showed that the total phenolics and flavonoids were 0.53506 ± 0.001335 mg GAE/g extract and 0.52067 ± 0.000731 mg QE/g extract, respectively. Meanwhile, the results of the antioxidant activity test with the FRAP method obtained a total antioxidant capacity of 29.85 μmol in the medium category and antioxidant activity with the DPPH method obtained an IC50 value of 597.0397 μg/mL in the very weak category.

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.

A non-marine horseshoe crab from the Middle Triassic (Anisian) of the Netherlands

Abstract Horseshoe crabs (Xiphosura) have a long evolutionary history starting in the Ordovician, but they have rarely been reported from the Netherlands. We report on the first Triassic horseshoe crab from the Netherlands identifiable to the species level, a specimen of the limulid Limulitella bronnii. We provide the first diagnosis for this species and refigure the holotype. The new specimen was found in the Middle Triassic (Anisian) Muschelkalk sediments of the Vossenveld Formation, in the Illyrian part of the stratigraphic profile of the Winterswijk quarry complex. The Winterswijk specimen represents the youngest occurrence of L. bronnii. The inferred non-marine habitat of this horseshoe crab species elsewhere in conjunction with occurrences of plant and insect remains within the same layer at Winterswijk suggest the specimen herein most probably did not live in marine conditions either. This species has previously been found in non-marine sediments in France and Germany, expanding its geographic range northward. Several faunal elements from Winterswijk including L. bronnii show resemblance to the roughly co-eval non-marine components of the Anisian Grès à Voltzia Formation in NE France, suggesting a paleobiogeographic connection between these regions in Western Europe.

Candidate Species for Florida Aquaculture: American Horseshoe Crab Limulus polyphemus

The American horseshoe crab, Limulus polyphemus, is a member of the phylum Arthropoda, subphylum Chelicerata, and class Merostomata. Although there are four horseshoe crab species worldwide, the American horseshoe crab is primarily found in the Atlantic Ocean along the East Coast of North America. The species ranges from Maine to Mexico, but is absent between Texas in the United States and Tabasco in Mexico. They mainly inhabit estuarine areas; however, juveniles and adults can venture into the shallow oceanic environments. Embryo development occurs in the intertidal zone of sandy beaches. Sandy or muddy bottom habitats are necessary for burrowing and benthic feeding. This publication will cover their natural history, aquaculture techniques, common diseases, and their uses and markets.

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.

Transcriptomic Signature of Horseshoe Crab Carcinoscorpius rotundicauda Hemocytes' Response to Lipopolysaccharides.

Carcinoscorpius rotundicauda (C. rotundicauda) is one of the four species of horseshoe crabs (HSCs). The HSC hemocytes store defense molecules that are released upon encountering invading pathogens. The HSCs rely on this innate immunity to continue its existence as a living fossil for more than 480 million years. To gain insight into the innate mechanisms involved, transcriptomic analysis was performed on isolated C. rotundicauda hemocytes challenged with lipopolysaccharides (LPS), the main components of the outer cell membrane of gram-negative bacteria. RNA-sequencing with Illumina HiSeq platform resulted in 232,628,086 and 245,448,176 raw reads corresponding to 190,326,253 and 201,180,020 high-quality mappable reads from control and LPS-stimulated hemocytes, respectively. Following LPS-stimulation, 79 genes were significantly upregulated and 265 genes were downregulated. The differentially expressed genes (DEGs) were related to multiple immune functional categories and pathways such as those of the cytoskeleton, Toll and Imd, apoptosis, MAP kinase (MAPK), inositol phosphate metabolism, phagosome, leucocyte endothelial migration, and gram-negative bacterial infection, among others. This study provides important information about the mechanisms of response to LPS, which is relevant for the understanding the HSCs' immune response.

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.

A new species of the Ordovician horseshoe crab Lunataspis

AbstractHorseshoe crabs as a group are renowned for their morphological conservatism punctuated by marked shifts in morphology associated with the occupation of non-marine environments and have been suggested to exhibit a consistent developmental trajectory throughout their evolutionary history. Here, we report a new species of horseshoe crab from the Ordovician (Late Sandbian) of Kingston, Ontario, Canada, from juvenile and adult material. This new species provides critical insight into the ontogeny and morphology of the earliest horseshoe crabs, indicating that at least some Palaeozoic forms had freely articulating tergites anterior to the fused thoracetron and an opisthosoma comprising 13 segments.

Carcinoscorpius rotundicauda (mangrove horseshoe crab)

The mangrove horseshoe crab, Carcinoscorpius rotundicauda, is one of the four extant species of horseshoe crabs. Mangrove horseshoe crabs have an important ecological role in the marine food chain, besides providing nutrient-rich eggs to migratory birds. However, their populations have been declining rapidly due to loss of habitat and irresponsible fishing. Hence, there is a need to understand their population-genetic structure and genetic diversity, which are essential for designing effective conservation strategies.

A A Trend of Zinc Uptake into Tachypleus Gigas Tissues After a Month of Exposure

Tachypleus gigas is one of the species of horseshoe crabs found in Peninsular Malaysia. Metals in the surrounding water can accumulate into horseshoe crab tissues. This study was conducted to determine zinc (Zn) uptake into horseshoe crab tissues after continuous exposure to the metals. T. gigas that were sampled from Gelang Patah, Johor and Cherating, Pahang were reared in the control tank and Zn treatment tank (20 mg/L) with aeration supply for a month. Twelve horseshoe crabs from Zn treatment tank and 6 horseshoe crabs from control tank were dissected for tissues namely operculum, gills, chelicerae, leg, digestive tract, hepatopancreas and carapace in different intervals (Day 0, Day 10, Day 20 and Day 30). These tissues were freeze dried and digested with 65% nitric acid on a hotplate at 200oC. Zn were measured using Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). Different concentrations of Zn were measured in different tissues. The tissues of T. gigas showed increased concentrations of Zn by following exposure in 30 days. The increasing pattern of Zn uptake into the tissues from day 0- 30 are clear. The gills showed highest concentrations of metals accumulation with the value of 6398.60 ± 909.51 µg/g (dw). Thus, uptake of Zn into T. gigas was confirmed, therefore it has the potential to become the suitable bio-indicator (especially their gills) to detect the metal pollution in the surrounding water.

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.

Development of methodology and reference intervals for the analysis of the free-ranging Atlantic horseshoe crab Limulus polyphemus hemolymph.

The Atlantic horseshoe crab, Limulus polyphemus, is valuable to fishing and biomedical industries, for education in public aquaria, and of ecological importance because the eggs provide a critical food source for migratory shorebirds. While hematologic and biochemical analyses are fundamental tools for health assessment, reports are limited for the four extant species of horseshoe crabs; Lpolyphemus (East Coast North America), Carcinoscorpius rotundicauda (mangrove horseshoe crab Southeast Asia), Tachypleus gigas, and Tachypleus tridentatus (Southeast and East Asia). This prospective study measured hemolymph values to determine RIs from free-ranging horseshoe crabs during the annual breeding aggregation in Delaware Bay, USA. Near-shore animals were briefly manually restrained for hemolymph collection from the cardiac sinus. Hematologic samples (25 males, 25 females) were collected for hemocytometer counts using the Natt and Herrick method. A second set of 50 animals (25 males, 25 females) was sampled for biochemistry measurands, including copper concentration by spectrophotometer, osmolality by vapor pressure osmometer, and protein electrophoresis. Total hemocyte counts varied widely (RI 6600-44300/µL). Electrolytes were consistent with osmoconformers living in seawater. There was a marked difference in sexes, with females showing much lower protein, glucose, and copper levels. Eleven females had multiple outliers, and all data were excluded from RI calculations. Further studies of this species outside of spawning season are needed. Expanding our knowledge of horseshoe crab health is important to decreasing morbidity and mortality in research applications and maximizing longevity and overall health in display settings.