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Endotoxin contamination in magnetic resonance imaging (MRI) contrast agents can pose a risk to patient safety causing immune reactions. Strict endotoxin limits are enforced for implants and catheters inserted into the body, but there are not clear rules for MRI contrast agents. Here, we investigated the efficacy of chromogenic LAL assay test for screening endotoxin activity in MRI contrast media manufactured in Malaysia. The powdered agent was dissolved in water for injection and endotoxin levels were measured. The coefficient of efficiency value for the standard curve, exhibiting r² ≥ 0.98, along with the absence of interfering substances and endotoxin activity below the regulatory threshold of 0.5 EU/mL, support the conclusion that the agent is unlikely to be pyrogenic or induce pyrogenic effect. [ABSTRACT FROM AUTHOR]

Atlantic horseshoe crabs (Limulus polyphemus) are prevalent in public aquarium touch pools. Despite their popularity, the literature concerning medical management under managed care is sparse. Noninfectious conditions include trauma to the exoskeleton and compound eyes; however, injury to the soft tissue connecting the telson and opisthosoma has not been previously reported. This report describes telson avulsion in three Atlantic horseshoe crabs at a public aquarium and attempted external stabilization with a vascular silicone tie in two of three affected animals. The horseshoe crab that received no veterinary intervention suffered a complete telson amputation 1 month after the initial injury. Although the two other horseshoe crabs did well postoperatively, and external stabilization prevented further avulsion or amputation, they still could not right themselves if flipped into dorsal recumbency. All three horseshoe crabs were ultimately euthanized due to animal welfare concerns. To avoid potentially serious telson ligament injury, horseshoe crabs should never be picked up by the telson. The authors recommend that telson ligament injuries be addressed promptly, any inciting cause be mitigated, and external stabilization be considered earlier. [ABSTRACT FROM AUTHOR]

Aim: Adult body size often exhibits patterns across large‐scale environmental gradients, creating ecogeographic clines. However, the form of body size clines varies across taxonomic groups, with linear and non‐linear patterns in body size observed in nature. Non‐linear body size clines have received less study, and questions remain about how environmental gradients interact to produce non‐linear clines. We examined the body size of the American horseshoe crab (Limulus polyphemus), a widely distributed marine arthropod, and evaluated the hypothesis that temperature and active season length can interact multiplicatively to result in a dome‐shaped distribution. Location: Fourteen states in the United States of America and three Mexican states, representing the entire geographic range of the species. Methods: We compiled environmental data and body size measurements from more than 49,000 individual horseshoe crabs. For each location, we extracted from the literature or calculated from raw data the mean male prosoma width and the mean female prosoma width. We applied a general additive modelling (GAM) approach to characterize the body size cline, test a hypothesis regarding temperature and season length, and explore evidence for the influence of additional environmental factors. Results: Model results indicate temperature and season length could act multiplicatively to produce dome‐shaped clines, and these findings align with and quantify previous anecdotal reports of a strong dome‐shaped body size cline across latitude for horseshoe crabs. Main Conclusions: Active season length appears to become relatively more influential on horseshoe crab body size in the northern part of their range, while temperature effects per se appear to dominate in southern latitudes. For horseshoe crabs, the pattern of size variation is consistent with the predictions of Optimal Resource Allocation models, but more study is needed to elucidate mechanistic underpinnings. Considering climate change projections, results from our study suggest future shifts in horseshoe crab body sizes. [ABSTRACT FROM AUTHOR]

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-β-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 β from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for β. 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 β subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and β subunits for BDG-dependent activation [ABSTRACT FROM AUTHOR]

Most fishermen in Mempawah Regency are fishing in demersal waters with the main target is shrimp which has a high economic value. However, the horseshoe crab as by catch is a marine protected animal. The aim of this study was to identify and analyze the abundance of horseshoe crab in Mempawah Mangrove Park (MMP) Waters. Sample was collected for 5 months, from April to August 2022, at MMP area. The fishing gear that was used for sampling is trammel net. Three species found in these waters are Tachypleus gigas, Tachypleus tridentatus, and Carsinoscorpius rotundicauda. Number of T. gigas was found is three, T. tridentatus was six, and C. rotundicauda was 30. Sex ratio of C. rotundicauda was dominated by male (24:6), then for T.tridentatus the number of male and female species was balanced (3:3). As for T. gigas, the female was dominant (1:2). The three species of horseshoe crab that found in MMP waters illustate that these waters are habitat, migration areas, or spawning ground for these species. C. rotundicauda was known to be the most found in mangrove areas and muddy substrates that support their population growth. [ABSTRACT FROM AUTHOR]

Limulidae (Horseshoe crab), or Mimi in the local name, are known; there are four species, namely Limulus polyphemus in North America and Asia, namely Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. In Indonesia, these species are protected by the Decree of the Minister of Forestry Number 12/KPTS-II/1987; meanwhile, illegal fishing still occurs. This study aims to identify Limulidae species and analyze Mimi's morphometric characteristics and distribution in North Sumatra to conserve the species. This research was conducted using a quantitative descriptive method in Leidong, Sei Berombang, Tanjung Tiram - North Sumatera, from September to December 2021. Biometric measurements were performed on 98 Mimi, and the data were statistically analyzed using the Kruskal Wallis test, Mann-Whitney test, and Multinomial logistics. The results showed that Mimi in the study area was identified as Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. The most commonly caught was T. tridentatus at the Tanjung Tiram. Based on the maximum width of the prosoma and body length, the largest to most petite sizes are T. tridentatus at Leidong (10.31 cm), T. gigas at Leidong, and Sei Berombang (10 cm) and C. rotundicauda at Sei Berombang (7.56 cm) respectively. The cluster analysis based on 27 morphometric characters showed that T. tridentatus had almost the same characteristics as T. gigas, while C. rotundicauda had its features. It can be concluded that the largest to most petite sizes of Mimi in the study area were T. tridentatus, T. gigas, and C. rotundicauda, respectively. [ABSTRACT FROM AUTHOR]

Horseshoe crabs are famous for their blood lysate that is extensively used for the detection of bacterial endotoxin contamination in injectable drugs and medical equipment. However, their existence is now threatened because of overexploitation for bleeding. Synthetic antimicrobial peptides or proteins created by bioengineering may be effective in solving the present predicament. Factor D (FD) is a bioactive substance that shows significant sensitivity to gram-negative bacteria. However, little is known about the expansion information of FD orthologs. In the present study, a total of nine FD orthologs were detected in horseshoe crabs according to the reference genomes. The composition of the FD orthologs in different species was highly conserved, although the common ancestor of the extant horseshoe crabs dates back to the Silurian period. These data suggested that the composition of FD orthologs in horseshoe crabs was formed before the speciation of these species. Considering that three rounds of whole-genome duplication (WGD) events may expanded the homologs in horseshoe crabs, while only one pair of FD orthologs showed significant collinearity according to the intraspecies comparative analysis. Unexpectedly, tandem duplication events that occurred before speciation also expanded the FD orthologs. In addition, several separately distributed FD orthologs also showed high conservation with each other. These data revealed that transposon-mediated duplication may have expanded the FD orthologs in horseshoe crabs. Intriguingly, the C-terminus of the FD orthologs is extremely conserved, although the expansion occurred more than 400 million years ago. In summary, our research provides evidence for the duplication of FD orthologs in horseshoe crabs. Additionally, by demonstrating the conservation of the segments in the C-terminus of these duplicated orthologs, our results also contributed to understanding the functional differentiation mechanism of the diversified FD orthologs. [ABSTRACT FROM AUTHOR]

There are six horseshoe crab marine protected areas (MPAs) in Guangdong Province. However, the baseline information on distribution, size and trend of horseshoe crab populations is virtually unknown. The lack of information has hindered the formulation of scientific management strategies and conservation actions. In this study, field surveys were conducted to investigate the current status of horseshoe crab populations in each of the six MPAs, and to analyze the macrobenthic communities in the intertidal zone. The results showed that juvenile horseshoe crabs were only found in Suixi Chinese horseshoe crab and Xuwen mangrove horseshoe crab MPAs. The abundance of the juveniles within Xuwen MPA was considerably low, i.e., only two juveniles were found. The abundance in Suixi MPAs was 6.45 ind·100 m–2, but the number of individuals in young size group was much lower compared to that of elder groups. The analysis of functional groups of macrobenthos in the MPAs intertidal areas demonstrated that those from Suixi were mainly plankton feeders, while those from Xuwen were primarily phytoplankton feeders. Plankton, microalgae, phytoplankton and other natural food sources are abundantly available, which can fulfill the feeding requirements of the juveniles. The population surveys within the horseshoe crab MPAs revealed the unsatisfactory situation of horseshoe crabs in Guangdong Province, and the management of these MPAs should be improved urgently. Conservation actions such as upgrading the protection level of the horseshoe crab, establishing scientifically-sound restocking program and effective management measures are imperatively required. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Over the past few decades, ocean hypoxia has been increasing due to human activities. Hypoxic stress, characterized by a reduced level of dissolved oxygen, is an escalating threat to marine ecosystems, with potentially devastating effects on the viability of endangered species such as the tri-spine horseshoe crab Tachypleus tridentatus. Even though this species is remarkably resilient to low oxygen levels, persistent hypoxia can negatively impact its population's survivability. The objective of this research was to examine the impact of short-term hypoxia on the behavior, gut microbiota, and metabolomics of juvenile T. tridentatus. First instar juvenile horseshoe crabs were subjected to hypoxic stress (2 mg O 2 /L) for 14 days and then recovered for 7 days in an environment with normal dissolved oxygen. The findings demonstrated that short-term hypoxia reduced the rates of swimming and burrowing of horseshoe crabs, and induced the change of tissue metabolites and intestinal flora malfunction. Additionally, in the hypoxia groups on day 14, 86 distinct metabolites showed a trend of downregulation, while 29 metabolites showed an upregulation trend. Arginine biosynthesis; histidine metabolism; vitamin B6 metabolism; aminoacyl-tRNA biosynthesis; and alanine, aspartate, and glutamate metabolism were the top five metabolic KEGG pathways (p < 0.05) enriched with 8 metabolites. In conclusion, our results provided new insights related to the behaviors, microbiota, and metabolites involved in juvenile T. tridentatus exposed to short-term hypoxic conditions and confirmed that hypoxia impairs their behavioral and physiological status., (© 2025 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Mercury (Hg) contamination is a major environmental concern that continuously impacts marine organisms, including the mangrove horseshoe crab (Carcinoscorpius rotundicauda). As a vulnerable species, C. rotundicauda holds significant ecological and evolutionary value, making it particularly susceptible to Hg pollution and deserving of focused conservation efforts. This study presents the first comprehensive histopathological and histochemical analysis of mercury toxicity in the digestive tract of adult C. rotundicauda. Through both qualitative and quantitative analyses of histopathological and histochemical alterations, we evaluated the effects of acute Hg exposure (0.5 mg/L and 5 mg/L) on the esophagus, stomach, and intestines over time. The results revealed significant dose- and time-dependent tissue damage, with the stomach exhibiting the most pronounced alterations, including epithelial detachment, vacuolation caused by fibers degradation in the loose connective tissue, and muscle layer disruption, followed by the esophagus and intestines. Additionally, mucous cell density in the hindgut submucosa decreased by 30 % after 96 h of acute Hg exposure (0.5 and 5 mg/L HgCl 2 ), indicating a weakened protective barrier. The tissue damage of the digestive tract may further have a negative impact on the health of the adult horseshoe crab, thus threatening the survival of C. rotundicauda population and reducing the biodiversity of the mangrove ecosystem. These findings underscore the critical threat posed by Hg pollution to the digestive system of C. rotundicauda and offer novel insights into the toxicological impact on marine invertebrates. Considering the tissue-damaging effects of Hg on adult horseshoe crabs, this study underscores the importance of regular monitoring of Hg contamination in natural habitats. The results offer valuable guidance for future habitat restoration and effective management of existing habitats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood‐coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200‐residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34‐residue, mostly unstructured pro‐peptide (PP) traverses, and thus blocks, the active‐site cleft of the CD in the opposite direction to a substrate. A central 'PP motif' (F35‐E‐G‐D‐I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an 'aspartate‐switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an 'activation segment'. Moreover, the mature N‐terminus is repositioned to penetrate the CD moiety and is anchored to a buried 'family‐specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin β and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal. [ABSTRACT FROM AUTHOR]

Introduction: Antimicrobial peptides (AMPs) are compounds with antimicrobial properties that are studied widely due to the development of resistance of pathogenic bacteria to antibiotics. In the present study, the toxicity and antimicrobial effects of two natural monomeric peptides (S3 and S∆3) were compared with S3-S∆3 hybrids and S3 tetramers. Material & Methods: Protein hybrids (S∆3S3-2mer-GS) S3-S∆3 and tetramer protein S3 (S3-4mer-GS) were expressed in E. coli. BL21 (DE3). Following that, the presence of mutant peptides was confirmed, and their antimicrobial activity was compared and evaluated with S3 and S∆3 monomers. Finally, the toxicity of tetramer and hybrid made on the MDA-MB-231 cell line was evaluated and compared. Findings: The toxicity of the hybrid was slightly increased, compared to the tetramer for eukaryotic cells; however, this increase was negligible at the active concentration of this protein. Cell survival for hybrids was lower for S3 and SΔ3; nonetheless, cell survival for each sequence decreased with increasing time. Furthermore, the inhibition of hybrid microbial growth was improved and compared with tetramer and S3-SΔ3. It was found that an increase in the positive charge of the hybrid protein did not have a toxic effect on the host bacteria. Discussion & Conclusion: Due to the appropriate expression and increased antimicrobial activity and negligible cytotoxicity, the hybrid peptide S3-S∆3 and tetramer S3 can be considered an effective production strategy to obtain AMPs.

Introduction: The study of the effects of synthetic peptides with antibacterial properties can provide more effective antibiotics. This study designed, expressed, and investigated the Sushi 3 tetramer peptide. Subsequently, it was compared in terms of changing antibacterial properties with another Sushi3 tetramer peptide the aspartic acid and proline amino acids of which were replaced with glycine and serine amino acids. Material & Methods: First, the mentioned Sushi3 tetramer peptide sequences were designed, constructed, and named Mer1 and Mer 2, respectively, and cloned separately into plasmid pET-26b (+) and finally transferred to E.coli BL21 host (DE3). After the expression of the peptides, the presence of peptides was confirmed by SDS-PAGE and Western blotting. Afterward, the antimicrobial activity of Mer1 and Mer 2 was evaluated and compared. Finally, the toxicity of the two tetramers made on the MDA-MB-231 cell line was evaluated and compared. Findings: Mer1 and Mer 2 had similar protein expression, and the toxic effect of both peptides on the cell line was not significantly different. however, Mer 2 had more effective antimicrobial effects than Mer1 at the same concentrations. Discussion & Conclusion: Evaluation of the effect of amino acid replacement with less negatively charge on increasing the antimicrobial activity of peptides is a suitable strategy. The above results increase the possibility of designing and producing antimicrobial peptides against antibiotic-resistant strains as the next generation of antibiotics.

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.

In order to compare the macrozoobenthic community and sedimentary environment with and without the presence of horseshoe crabs, the benthic macrofauna, sediment grain size, chromium (Cr), cobalt (Co), nickel (Ni), polycyclic aromatic hydrocarbon (PAHs), organic carbon and nitrogen were seasonally investigated at site A (with horseshoe crab presence), sites B and C (without horseshoe crab presence) in the Crocodile Island intertidal zone in Xiamen from June 2018 to July 2019. The results showed that most of the community parameters of benthic macrofauna, population parameters of common benthic macrofauna and environmental parameters were significantly different at the sites with horseshoe crab versus non-horseshoe crab sites. A two-way ANOVA test showed that the densities of Ceratonereis erythraeensis and Sigambra hanaokai had significant site variation. Cluster and non-metric multi-dimensional scaling (NMDS) analysis showed the community composition of benthic macrofauna was significantly different among the three sampling sites over four seasons. The mean sand content at site A (64.32%) was higher than those at site B (36.01%) and site C (18.86%). Conversely, the mean contents of silt, clay, Cr, Co, Ni, organic carbon, organic nitrogen, phenanthrene, and pyrene at site A were lower than those at site B and site C. These observations are consistent with the expected preferences of horseshoe crabs to live in areas with 60% sand content, which is associated with abundant and edible clamworms in the Crocodile Island intertidal zone, Xiamen. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) are ubiquitous in marine sedimentary environments. Their occurrence in horseshoe crabs and interactions with the sedimentary environment has not been determined. In this study, MPs, mostly microfibers, were found in all extracted gastrointestinal tract (GIT) samples of the juvenile Tachypleus tridentatus from the northern Beibu Gulf, China. The MP concentrations (4–53 items ind−1) were higher than those in most marine benthic invertebrates (< 15 items ind−1) reported in Chinese waters, despite their generally low level in habitat sediments (9-1818 items kg−1). The correlation between the juveniles and sediments was not evident, but the abundance in juvenile horseshoe crab GITs decreased with ages. The findings were relevant to the life-history characteristics of the species, typically with limited dispersal capability and their spending nine years or longer time living in mangrove wetlands during low tides, with apparent ontogenetic changes in their diets. These baseline data enable a better understanding of MP availability in benthic macroinvertebrates, and the ecological risks present in the ecosystems. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

An exceptionally preserved specimen of the horseshoe crab Euproops danae (Xiphosurida, Belinurina) in a siderite concretion from the Carboniferous (Upper Pennsylvanian, Virgilian) Lawrence Formation, Kansas, shows anatomical details of the prosomal musculature. The extrinsic appendicular muscles are comparable to those of Limulus polyphemus (the modern American horseshoe crab), demonstrating anatomical conservatism within Xiphosurida that spans two morphologically disparate subgroups, Belinurina and Limulina. The three‐dimensional preservation of muscles highlights how siderite concretion fossils (including those of the Mazon Creek Konservat‐Lagerstätte) have better preservational fidelity than previously realized and the potential to reveal new anatomical information, especially with regard to the labile tissues of euarthropods. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A transcriptomic study of Tachypleus gigas hemocytes after 1-h LPS stimulation. • A total of 2287 genes were differentially expressed upon LPS stimulation. • Multiple immune-related genes and pathways were differentially regulated. • Genes associated with cancer and infection in vertebrates were also modulated. [ABSTRACT FROM AUTHOR]

We used citizen science to improve understanding of population trends and behavior in the American horseshoe crab ('Limulus polyphemus') along the entire coast of Florida. First, we used 18 years of public sightings data, beginning in 2002, on horseshoe crab mating to determine which spawning locations were used most. Then, a subset of those locations was more rigorously surveyed through the Florida Horseshoe Crab Watch program. Florida Horseshoe Crab Watch, implemented in 2015 by a collaboration between the Florida Fish and Wildlife Conservation Commission (FWC), University of Florida Institute of Food and Agricultural Science (UF/IFAS), University of Florida Department of Biology (UF), and Florida Sea Grant (FSG), uses trained citizen volunteers to survey beaches with high spawning activity. Volunteers count, weigh, measure, and tag horseshoe crabs that are nesting on the shoreline. These data contribute to a nationwide mark–recapture program managed by the United States Fish and Wildlife Service (USFWS) and are used in Florida to inform species management decisions and to increase general understanding of the species. We developed and adapted both phases of the research by implementing modern technology, improving survey design, and expanding geographic coverage. The quality and accuracy of public reports have improved with technological advancements. The quality of the morphological data collected by Florida Horseshoe Crab Watch volunteers was comparable to that of data collected by professional scientists. Our design can serve as a model for programs regardless of region or taxon of interest and when funding is limited but public interest is high.

Horseshoe crabs within Austrolimulidae represent the extreme limits to which the xiphosurid Bauplan could be modified. Recent interest in this group has uncovered an unprecedented diversity of these odd-ball xiphosurids and led to suggestions that Austrolimulidae arose during the Permian Period and had become extinct by the end of the Triassic Period. Here, we extend the temporal record of Austrolimulidae by documenting a new horseshoe crab from the Lower Jurassic (Hettangian) Bayreuth Formation, Franconiolimulus pochankei gen. et sp. nov. The novel specimen displays hypertrophied genal spines, a key feature indicative of Austrolimulidae, but does not show as prominent accentuation or reduction of other exoskeletal sections. In considering the interesting family, we explore the possible origins and explanations for the bizarre morphologies exhibited by the Austrolimulidae and present hypotheses regarding the extinction of the group. Further examination of horseshoe crab fossils with unique features will undoubtedly continue to increase the diversity and disparity of these curious xiphosurids. [ABSTRACT FROM AUTHOR]

Horseshoe crabs are archetypal marine chelicerates with an exceptionally long fossil record. Due to the historical nature of the genus Limulus, which extends back to Linnaeus' descriptions, many horseshoe crab fossils were traditionally placed in Limulus and the family Limulidae. Despite continued research into the accurate placement of species within Limulidae, a systematic outline of characteristics that define limulid genera, specifically using exclusively dorsal characteristics, does not yet exist. However, such an approach is essential as appendage data is rarely preserved in fossil horseshoe crabs. Here we present a systematic review of Limulidae with a focus on dorsal features, and illustrate all accepted limulid species across the 12 genera. Through this descriptive lens, we consider the validity of supposed Limulus species outlined in a recent xiphosurid review. We find evidence for only one fossil Limulus species: Limulus coffini. This revision therefore excludes Limulus from Jurassic‐aged deposits. We refer 'Limulus' darwini from the Upper Jurassic (Upper Tithonian) of Poland to Crenatolimulus darwini comb. nov. and 'Limulus' woodwardi from the Middle Jurassic (Aalenian) of England to Mesolimulus woodwardi comb. nov. This highlights that the Limulus evolutionary record is highly constrained and started as recently as the Late Cretaceous. The rare Limulus fossil record emphasizes the current need for conservation of extant species and the importance of thoroughly scrutinizing the morphology of fossil specimens to uncover all facets of the limited limulid evolutionary record. [ABSTRACT FROM AUTHOR]

The capacity of organisms to change their sex has evolved independently in several species. Sex change has been widely studied, but research approaches have differed and conclusions have often been specific. Although sex allocation theory provides a unifying framework for the study of sex change, this unity has not always been appreciated, Environmental sex reversal (ESR), which results in a mismatch between genotypic and phenotypic sex, is well documented in numerous species and may be induced by chemical exposure. In the present study, an attempt was made to hypothesize the phenomenon of sex reversal in both Asian species (Tachypleus gigas and Carcinoscorpius rotundicauda) of the horseshoe crab in accordance with the size of the animals. Our findings suggest that both Asian species of horseshoe crabs ultimately show a positive sign of sex reversal phenomenon, however, this hypothesis needs further confirmation by studying the hormonal changes at all moulting stages of the horseshoe crab. [ABSTRACT FROM AUTHOR]

Horseshoe crab population sizes and trends have been previously studied using surveys of spawning adults and counts of eggs from surface (top 5 cm) and deep (20 cm) sediment samples. The correlations between surface and deep eggs were studied at two locations, Delaware Bay and Jamaica Bay, USA, and the correlations between egg densities and spawning counts were examined in Jamaica Bay.There were significantly higher densities of eggs in deep sediments than in surface sediments. Only about 10% of the variability in surface egg density was explained by deep egg density. The numerical patterns between surface and deep eggs were similar between Delaware Bay and Jamaica Bay and across sampling dates.Nearly 20% of the deep samples in the combined data from Delaware Bay and Jamaica Bay with an egg density of ≥100,000 m−2 had zero surface eggs. Therefore, the use of surface eggs as an indicator of habitat suitability and spawning intensity may seriously underestimate the importance of a beach for spawning horseshoe crabs.When paired with nearest survey date, Jamaica Bay spawning indices did not predict deep or surface egg densities. This may be related to a temporal mismatch between survey methods, the extreme overdispersion (patchiness) of the eggs, and/or the dynamics of egg distribution after exhumation.Both egg density and spawning surveys can provide useful data on habitat suitability for horseshoe crabs and can offer excellent opportunities for student and citizen scientist engagement. More labour is required for egg surveys than spawning surveys because of the time required to sample, sort, and enumerate the eggs. [ABSTRACT FROM AUTHOR]

The mangrove horseshoe crab, Carcinoscorpius rotundicauda, has divergent populations between the east and west coasts of Peninsular Malaysia, with the southern coast acting as a land barrier. The actual position of such a genetic break along Peninsular Malaysia as well as the connectivity status of the southernmost C. rotundicauda populations with east and west coast populations remain unexplored, however.The aim was to investigate the genetic diversity and structure of C. rotundicauda populations from the west (Kuala Sepetang in State Perak), east (Balok in State Pahang), and southern (Pendas in State Johor) coasts of Peninsular Malaysia. Haemolymph samples from adult C. rotundicauda specimens (n = 152) and eggs from their freshly deposited nests (n = 190) were collected monthly (from January 2016 to January 2017) for the sequencing of mitochondrial cytochrome c oxidase subunit I (COI).Gene isolates of C. rotundicauda from the present study were compared with National Center for Biotechnology Information (NCBI) GenBank sequences to cover most of the range of the species in Asia. A neighbour‐joining tree strongly supported two clades, separating the west‐coast populations from the south‐ and east‐coast populations, with further substructure patterns.Both haplotype network and barrier analyses revealed a genetic break within the Strait of Malacca instead of the southern tip of Peninsular Malaysia. The southernmost samples from the Strait of Johor formed a haplotypic diverse gene pool that appeared only as a subclade of the eastern populations. In a detailed haplotype network of 347 individuals, individuals with similar COI sequences indicate connectivity between C. rotundicauda on the east and C. rotundicauda on south and south west of Peninsular Malaysia.Overall, the genetic break between C. rotundicauda populations is better explained by the convergent ocean currents and available mangrove habitats on the west coast (i.e. Strait of Malacca), rather than the point of the Malay Peninsula acting as a land barrier alone. [ABSTRACT FROM AUTHOR]

The Tachypleus tridentatus (Chelicerata: Xiphosura) is an arthropod that usually displays high site fidelity by restricting its distribution to natal vicinities. Briefly, shore perturbation from boating (Kudat), electric, cyanide and bombing (Kunak) and tourism (Semporna) in Sabah can impair the T. tridentatus growth and produce sexually mature adults with unusual size. The 8-point morphometry of Tachypleus tridentatus from Kudat (Sulu Sea), Kunak and Semporna (Celebes Sea), produces the final output constituting accurate size classification for the species. Meanwhile, T. tridentatus are sexually dimorphic (p = 0.968-0.989), where male T. tridentatus (from Kudat) has prosoma width (27.75 ± 2.68 cm) and weight (1050 ± 610 g) in smaller ranges when compared to the female (33.27 ± 4.68 cm; 3020 ± 1480 g) using the same comparison. In addition, the T. tridentatus populations from Kunak (prosoma width: male = 28.91 ± 1.48 cm, female = 29.44 ± 5.47 cm; weight: male = 800 ± 275 g; female = 2550 ± 155 g) and Semporna (prosoma width: male = 21.73 ± 1.34 cm, female = 24.42 ± 1.36 cm; weight: male = 485 ± 306 g, female = 1320 ± 640 g) differed site-wise. The descriptive statistics (average and standard deviation) relate 7.1-23.0% of T. tridentatus with negative size class. However, the applied statistics using stepwise analysis and regression curve (r2 = 0.566-0.833), relates 30.4% of T. tridentatus with negative size class. Considering weight to produce 100% T. tridentatus with positive size class, it is biased because some crabs are gravid with eggs and if attained unpaired (male and female), they may have recently fed. Therefore, the prosoma (male), interorbit (female) and telson lengths are identified as most appropriate for the size classification of T. tridentatus. Thus, the findings provide a novel baseline for conservation studies that monitor symmetrical and unusual growth in the T. Tridentatus wild stocks. [ABSTRACT FROM AUTHOR]

The Limulus amebocyte lysate (LAL) isolated from cells in the horseshoe crab (HSC) hemolymph is a critical resource for global biomedical and pharmaceutical quality control and sterility testing. Given the necessity of and limitations associated with wild capture, a conservational approach to LAL harvesting would benefit the medical community that relies on the raw material while helping ensure species viability. We posited that aquaculture and year-round collection represented a sustainable alternative for the production of LAL from a finite HSC cohort, thereby averting the impact of current practices on wild populations. Given the specter of captivity diseases linked to diet, such as panhypoproteinemia, this work, at the outset, focused on optimizing a feed formulation to ensure animal vitality. In turn, each preparation required evaluation with respect to effects on LAL, as well as vital HSC health markers, so as to meet or exceed industry requirements and establish a new supply chain paradigm. In this controlled husbandry study, we conducted three 8-week feeding trials and demonstrated a ∼7-fold LAL reactivity range among the HSC feed groups. Relative protein abundance patterns of HSC amebocyte clotting factors (i.e., Factor C, Factor B, and proclotting enzyme) were influenced by diet in particular, and the up-regulation of specific LAL factors correlated with enhanced reactivity. These results also cite the discovery that coagulation Factor C, the LPS-sensitive serine protease proenzyme, may be a phosphoprotein.

Horseshoe crabs are an iconic group of extant chelicerates, with a stunning fossil record that extends to at least the Lower Ordovician (~480 million years ago). As such, the group has retained significant biological and palaeontological interest. The sporadic nature of descriptive and systematic research into fossil horseshoe crabs over the last two centuries has spread information on the group across more than 200 texts dating from the early nineteenth century to the present day. We present the most comprehensive pictorial atlas of horseshoe crabs to date to pool these important data together. This review highlights taxa such as Bellinurus lacoei and Limulus priscus that have never been documented with photography. Furthermore, key morphological features of the true horseshoe crab (Xiphosurida) families—Austrolimulidae, Belinuridae, Limulidae, Paleolimulidae, and Rolfeiidae—are described. The evolutionary history of horseshoe crabs is reviewed and the current issues facing any possible biogeographic work are presented. Four major future directions that should be adopted by horseshoe crab researchers are outlined. We conclude that this review provides the basis for innovative geographic and geometric morphometric studies needed to uncover facets of horseshoe crab evolution.

This study investigated the effects of prolonged starvation on oxygen consumption, ammonia-N excretion, and blood chemistry of the horseshoe crab, Carcinoscorpius rotundicauda. Starvation over a period of 7 weeks showed no significant difference of body weight between the starved and fed groups. The oxygen consumption rate decreased during weeks 1-4 and then significantly increased after week 6 of starvation. Starvation also resulted in a significant increase in ammonia-N rate from week 3 to 7. The O:N ratios were significantly reduced in the starved group from week 3 to 7. Starving induced the reduction of hemolymph osmolality from week 5. Hemolymph Na+ and Cl‾ of the starved group decreased from week 4 for Na+ and from week 3 to 7 for Cl‾, while hemolymph K+ increased from week 4 to 7. Hemolymph K+ of both groups was hyperionic during the experiment. Thus, horseshoe crab can survive starvation for more than 7 weeks.

The population of horseshoe crabs, Tachypleus gigas, in Malaysian waters is declining due to overexploitation, pollution, and loss of natural habitats. As an initiative to increase their population, adult T. gigas were reintroduced in Setiu lagoon, Terengganu. In the interest of sustainable management of bivalve resources in that area, feeding experiments were carried out to determine quantity consumption and feeding preferences of adult T. gigas in captivity on four species of economically important bivalves from Setiu namely Scapharca cornea, Meretrix meretrix, Geloina expansa and Magallana bilineata. Tachypleus gigas consumed less than a single intact bivalve per day and the amount increased to almost twice when fed with bivalve flesh. Females T. gigas showed a preference for intact M. meretrix, whereas males showed preference for M. bilineata and M. meretrix when fed with flesh only. Our findings suggest that T. gigas may not prey upon live adult bivalves in Setiu as they only scavenge on dead or weak bivalves. However, the impact of horseshoe crab reintroduction on other small invertebrates in Setiu including the juveniles of these bivalves needs to be explored in order to ensure the success of this conservation effort. [ABSTRACT FROM AUTHOR]

We used citizen science to improve understanding of population trends and behavior in the American horseshoe crab (Limulus polyphemus) along the entire coast of Florida. First, we used 18 years of public sightings data, beginning in 2002, on horseshoe crab mating to determine which spawning locations were used most. Then, a subset of those locations was more rigorously surveyed through the Florida Horseshoe Crab Watch program. Florida Horseshoe Crab Watch, implemented in 2015 by a collaboration between the Florida Fish and Wildlife Conservation Commission (FWC), University of Florida Institute of Food and Agricultural Science (UF/IFAS), University of Florida Department of Biology (UF), and Florida Sea Grant (FSG), uses trained citizen volunteers to survey beaches with high spawning activity. Volunteers count, weigh, measure, and tag horseshoe crabs that are nesting on the shoreline. These data contribute to a nationwide mark--recapture program managed by the United States Fish and Wildlife Service (USFWS) and are used in Florida to inform species management decisions and to increase general understanding of the species. We developed and adapted both phases of the research by implementing modern technology, improving survey design, and expanding geographic coverage. The quality and accuracy of public reports have improved with technological advancements. The quality of the morphological data collected by Florida Horseshoe Crab Watch volunteers was comparable to that of data collected by professional scientists. Our design can serve as a model for programs regardless of region or taxon of interest and when funding is limited but public interest is high. [ABSTRACT FROM AUTHOR]

In this paper, horseshoe crab exoskeleton and exuviae were collected on the coasts of Yucatan and physicochemical studies were carried out. Samples from different sections of the exoskeleton were taken to evaluate chitin content and properties in order to be compared with exuviae. The isolation of chitin was realized by chemical processes involving deproteinization, demineralization and discoloration. Physicochemical properties of chitin extracted from both sources were compared in terms of characterization analysis in particular achieved under EDX, FTIR and calorimetric techniques (DSC and TGA). In such comparative analysis lies the importance to establish differences or similitudes in chitin contents, structure, etc. Scanning Electron Microcopy was conducted on exuviae and exoskeleton selected parts in order to identify horseshoe crab structure and surface morphology. Differential scanning calorimetry was focused on changes in physical and chemical structures at different samples showing two endothermic peaks related to the complete elimination of hydroxyl groups attached to polysaccharide rings. TGA thermograms were characterized by endo and exotherms corresponding to water evaporation and decomposition of polymer respectively. Exoskeleton SEM images shown a tubular network and porous surface while exuviae exhibited irregular surface and diatom unicellular microalgae embedded. Calorimetric measurements confirmed that exuviae is richer in chitin than exoskeleton and that the main allotropic crystal structure corresponds to α‐chitin. FTIR analysis supported these results by exhibiting different absorption bands corresponding to α‐chitin. [ABSTRACT FROM AUTHOR]

Horseshoe crabs have survived for a long period due to their myriad defence mechanism. Thorough studies were conducted on their adaptive defence mechanisms but few highlighted their primary innate defence. This study aimed to determine the antifouling properties of the methanolic crude extracts and fractions obtained from the book gills and carapace of the Tachypleus gigas and to characterize their bioactive metabolites, using the detachment and preventive measurements. The active fractions recovered from the recycling HPLC were further analysed using GC-MS and major active constituents were identified. The results obtained indicated that the extract and fractions of T. gigas had significant detachment mode of actions for antifouling activities. Chemical interactions such as synergistic effects were observed in both the book gills and the carapace. Chemical profiles indicated the presence of organic acids such as amino acid, simple sugars and fatty acids. The observed activities might be caused by nutrient-induced biofilm dispersion mechanisms. These findings may provide new insight into the defence mechanism of the horseshoe crabs against biofilm formation. The isolated active fractions and the identified molecules from the horseshoe crab T. gigas may be applied as a dispersal antifouling agents. Further studies are needed to confirm these prospective potential applications. [ABSTRACT FROM AUTHOR]

Several aquatic species use beach foreshores to lay their eggs. Understanding the dynamics of egg entrainment versus egg retention in foreshore sediment is important to delivering eggs to foraging species, and to predicting fecundity and population responses of beach nesting species to harvest and habitat management. Exhumation of these eggs is primarily by bioturbation and wave action. This study isolates effects of wave and swash processes on exhumation, entrainment, and transport of horseshoe crab eggs by conducting a 1-day field study designed to avoid effects of bioturbation by spawning and migratory shorebird predation. Dyed sand and horseshoe crab (Limulus polyphemus) eggs were injected into the foreshore of an estuarine beach in Delaware Bay, USA and sampled during 110 swash events using streamer traps. Significant wave heights ranged from 0.30 to 0.47 m. The quantity of eggs exhumed from the mid-foreshore by waves and swash was 3.8 times greater than from the upper foreshore where activation was by swash alone. Swash flows were skewed offshore, but egg transport was predominantly onshore from bore collapse. Egg release was greatest in early rising tide, making this the most productive time for shorebird foraging. Release of eggs from mid-foreshore may be dominated by wave action during high energy days with little spawning or by bioturbation by horseshoe crabs during intensive spawning. Activation depths in the swash were low, indicating that eggs buried on the upper foreshore can reach later stages of development after spawning ceases. [ABSTRACT FROM AUTHOR]