Horseshoe Crab Research Articles

Morphometry of Limulidae (Oscar E. Brown) in Leidong, Sei Berombang, and Tanjung Tiram, North Sumatera

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.

The Mighty Bite by Nathan Hale (review)

Reviewed by: The Mighty Bite by Nathan Hale Ally Byerly Hale, Nathan The Mighty Bite; written and illus. by Nathan Hale. Amulet/Abrams, 2023 [288p] Trade ed. ISBN 9781419765537 $14.99 E-book ed. ISBN 9781647008758 $13.49 Reviewed from digital galleys R Gr. 1-4 Trilobite (an ancient arthropod which falls between a horseshoe crab and a rolypoly) lives happily and humbly on the Island of Losta Founda, a land populated by prehistoric and mythic creatures, until a vivid dream ignites his hunger for fame and fortune. Unfortunately, Trilobite and his companion, Amber the Ambulocetus (an amphibious whale that resembles a crocodile), don't quite know how they'll go about finding fame (an attempt at singing goes terribly awry) until they encounter Tiffany Timber, a paleo-newscaster who wields a taser and a tablet in her hunt for the latest scoop. Trilobite and Amber are quickly drawn into the absurdities of Timber's newscaster world, stumbling from one episodic misadventure to the next and facing everything from a claymation smear campaign to an interview with a [End Page 260] sea cucumber in their quest for fame, fortune, and journalistic integrity. The story's premise is as creative as it is absurd, and the farcical volume is structured like a series of daily strips, replete with silly gags and wry one-liners. Hale's dynamic layouts guide the eye to create a quick, accessible read, and perspective and transitions are skillfully used to produce an immersive, cinematic experience that blurs the boundaries between space, time, and genre. Hand-drawn, heavily cross-hatched illustrations brilliantly juxtapose with the modern video interface of the fame-hungry protagonists without privileging one medium over the other. Instead, the narrative plays on the commonalities between the sequential art forms—from the framing to the timing to the editing, and ultimately pays homage to the timeless craft of storytelling. This will be an absolute treat for any reader who likes their natural history with a side of whimsy. Copyright © 2023 The Board of Trustees of the University of Illinois

The use of physiological and transcriptional analyses to examine molting regulatory mechanisms in juvenile horseshoe crab Tachypleus tridentatus

Molting is a necessary developmental process in crustaceans, and is holistically regulated by a range of hormones and associated genes. In order to investigate physiological changes occurring at the molecular level during the molting cycle of juvenile Tachypleus tridentatus, chitinase and N-acetyl-β-D-glucosaminidase (β-NAGase) activities were firstly measured by absorbance spectrophotometry. In addition, ecdysone content was quantified by ELISA, and changes in expression levels of genes relevant to ecdysis were examined based on quantitative real-time PCR (qRT-PCR) and RNA-sequencing (RNA-seq). Results showed that chitinase, β-NAGase activity, and ecdysone content increased at the intermolt stages, peaked at premolt stages, and decreased to a lower level at molt and postmolt stages. Chitinase and β-NAGase were positively correlated with ecdysone. RNA-seq revealed that a total of 6482 differentially expressed genes (DEGs) in the four different molting stages, and 5763 DEGs had higher expression levels in the premolt stage than in the intermolt stage. A total of 59 shared DEGs were identified in all molting stages, and the highly expressed DEGs were mainly related to chitin binding, stratum corneum structural composition and neuropeptide hormone activity signaling pathways. KEGG pathway analysis showed that at the premolt stage, digestion, transport, and catabolic signaling pathways were significantly enriched and innate immune response pathways were significantly enriched as molting progressed. These results suggested that the molting of juvenile horseshoe crabs may be directly regulated by ecdysone, and chitinase and β-NAGase play a synergistic role with ecdysone during the molting cycle. The key biological processes and signaling pathways involved in hormone regulation, epidermal structural proteins, catabolism, apoptosis, and immune response potentially regulate the molting process of horseshoe crab juveniles. This study provides a theoretical basis for enhancing understanding of the molting and growth processes in marine arthropods, and a strong theoretical support for the developmenting of captive rearing technologies for Chinese horseshoe crabs.

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.

Extraction and Purification of Bioactive Peptide with Antimicrobial Properties from Horseshoe Crab

Abstract: Peptides are small biomolecules that act as bioactive or host defense molecules. These peptides have a significant role in medicine because of their broad spectrum antimicrobial properties. Marine sources are the largest supply of peptides and natural small molecules since they represent half of the total global biodiversity. Among the different crab species, the horseshoe crab is widely used for extracting the peptides. The horseshoe crab is a marine and brackish water arthropod; it plays a crucial role in the reel and whelk fishing industry and the production of important pharmaceutical bioactive components. This review highlights the details of the extraction and purification of antimicrobial peptides from the horseshoe crab. These peptides represent trends and opportunities for the future, with a focus on the pharmaceutical industries.

Effects of Ca2+ ions on the horseshoe crab coagulation cascade triggered by lipopolysaccharide.

The lipopolysaccharide (LPS)-triggered horseshoe crab coagulation cascade is composed of three protease zymogens, prochelicerase C (proC), prochelicerase B (proB) and the proclotting enzyme (proCE). In this study, we found that Ca 2+ ions increase the production of the clotting enzyme as a result of a cascade reaction reconstituted by recombinant proteins of wild-type (WT) proC, WT proB and WT proCE. We divided the cascade into three stages: autocatalytic activation of WT proC on the surface of LPS into WT α-chelicerase C (Stage 1); activation of WT proB on the surface of LPS into WT chelicerase B by WT α-chelicerase C (Stage 2) and activation of WT proce into WT CE by chelicerase B (Stage 3). Ca2+ ions enhanced the proteolytic activation in Stage 2, but not those in Stages 1 and 3. Moreover, we performed isothermal titration calorimetry to clarify the interaction of LPS or the recombinant zymogens with Ca2+ ions. LPS interacted with Ca2+ ions at an association constant of Ka= 4.7 × 104M-1, but not with any of the recombinant zymogens. We concluded that LPS bound with Ca2+ ions facilitates the chain reaction of the cascade as a more efficient scaffold than LPS itself.

Towards effective wildlife protection law for marine species in China: A stakeholders’ perspective

IntroductionAs one of the megadiverse countries, the effectiveness of wildlife protection in China is of great significance to global biodiversity conservation. With continued evolution and revisions, China’s Wildlife Protection Law has listed over 140 marine species; however, it is still inclined toward terrestrial animals.MethodsTo narrow the gap between compliance and enforcement, we collected 1,309 effective responses from various coastal cities of China through an anonymous online questionnaire survey, to investigate their exposure, understanding and attitudes toward Wildlife Protection Law for marine species (mWPL).ResultsMost respondents demonstrated an overall good understanding about the context, necessity and effectiveness of mWPL. The fisher communities were found to be more aware of the dissemination and implementation of mWPL. However, they understood less of the penal system, and exhibited negative attitudes toward the necessity and punishment of the legislation, probably due to the conflicts between resource utilizations and legislative interventions. The participants also indicated that seahorses, horseshoe crabs and corals were commonly subjected to illegal exploitations.DiscussionWhile most respondents suggested greater fines, tighter laws and better public enforcement, we advocate the exploration of bottom-up options such as community engagement and environmental education to improve compliance and implementation of mWPL for the benefit of marine wildlife conservation in China.

Dual Functions of labial Resolve the Hox Logic of Chelicerate Head Segments.

Despite an abundance of gene expression surveys, comparatively little is known about Hox gene function in Chelicerata. Previous investigations of paralogs of labial (lab) and Deformed (Dfd) in a spider have shown that these play a role in tissue maintenance of the pedipalp segment (lab-1) and in patterning the first walking leg identity (Dfd-1), respectively. However, extrapolations of these data across chelicerates are hindered by the existence of duplicated Hox genes in arachnopulmonates (e.g., spiders and scorpions), which have resulted from an ancient whole genome duplication (WGD) event. Here, we investigated the function of the single-copy ortholog of lab in the harvestman Phalangium opilio, an exemplar of a lineage that was not subject to this WGD. Embryonic RNA interference against lab resulted in two classes of phenotypes: homeotic transformations of pedipalps to chelicerae, as well as reduction and fusion of the pedipalp and leg 1 segments. To test for combinatorial function, we performed a double knockdown of lab and Dfd, which resulted in a homeotic transformation of both pedipalps and the first walking legs into cheliceral identity, whereas the second walking leg is transformed into a pedipalpal identity. Taken together, these results elucidate a model for the Hox logic of head segments in Chelicerata. To substantiate the validity of this model, we performed expression surveys for lab and Dfd paralogs in scorpions and horseshoe crabs. We show that repetition of morphologically similar appendages is correlated with uniform expression levels of the Hox genes lab and Dfd, irrespective of the number of gene copies.

Impact assessment of human activities on resources of juvenile horseshoe crabs in Hainan coastal areas, China

The booming coastal zone economy poses increasing anthropogenic threats to marine life and habitats. Using the endangered living fossil horseshoe crab (HSC) as an example, we quantified the intensity of various anthropogenic pressures along the coast of Hainan Island, China, and for the first time assessed their impact on the distribution of juvenile HSCs through a field survey, remote sensing, spatial geographic modeling, and machine learning methods. The results indicate that the Danzhou Bay needs to be protected as a priority based on species and anthropogenic pressure information. Aquaculture and port activities dramatically impact the density of HSCs and therefore be managed priority. Finally, a threshold effect between total, coastal residential, and beach pressure and the density of juvenile HSCs were detected, which indicates the need for a balance between development and conservation as well as the designation of suitable sites for the construction of marine protected areas.

Estimating recruitment rate and population dynamics at a migratory stopover site using an integrated population model

AbstractConsideration of the full annual cycle population dynamics can provide useful insight for conservation efforts, but collecting data needed to estimate demographic parameters is often logistically difficult. For species that breed in remote areas, monitoring is often conducted during migratory stopover or at nonbreeding sites, and the recruitment rate of new breeding adults can be difficult to estimate directly. Here, we present an integrated population model that uses mark‐resight and count data to estimate survival probability, population growth rate, and recruitment rate for an Arctic‐breeding shorebird of conservation concern, the red knot (Calidris canutus rufa), from data collected during spring stopover in Delaware Bay, USA, from 2005 to 2018. At this site, red knots feed primarily on the eggs of spawning horseshoe crabs (Limulus polyphemus), a legally harvested species. We used this model to estimate the relationship between horseshoe crab abundance and red knot demographics, which informed a recent revision to the framework used to establish horseshoe crab harvest regulations. Our analysis indicates that the red knot population was most likely stable from 2005 to 2018 (average λ = 1.03, 95% credible interval [CRI]: 0.961, 1.15) despite low recruitment rates (average ρ = 0.088, 95% CRI: 0.012, 0.18). Adult survival probability was positively associated with horseshoe crab abundance in the same year (β = 0.35, 95% CRI: 0.09, 0.63), but we found no effect of horseshoe crab abundance two years previously on recruitment of new adults (β = −0.08, 95% CRI: −0.41, 0.38). Our approach demonstrates the utility of integrated population models for understanding population dynamics, even when data are only available from migratory stopover monitoring.

Life on a lake bottom: diverse horseshoe crab and fish ichnofauna from Yanchang Group (Upper Triassic) of northern Shaanxi Province, China–Implications for palaeoenvironment

ABSTRACT An ichnoassemblage comprising trackways and trails of horseshoe crabs and fishes has been discovered in the Upper Triassic Wayaobu Formation of the Ordos Basin, Shaanxi Province, northern China. This is a lacustrine coal-bearing succession with sandstones, mudstones and oil shales. Two trace-bearing beds are present, one with fine sandstone, the other being a mudstone layer. The traces are assigned to Kouphichnium isp., different Undichna ichnospecies representing U. bina, U. britannica, U. ?quina, U. unisulca and U. isp., and to an indeterminate trackway. Kouphichnium isp. shows long trackways dominated by impressions of the pusher legs, often accompanied by a telson trace. While these were probably left by bottom walking horseshoe crabs, others showing elongated curved scratches lacking a telson trace might have been left by swimming individuals. Remarkable is the large size of the Kouphichnium trackmaker if compared to known Triassic body fossils. The ichnofauna is described and the ichnotaxonomy of Undichna and similar forms discussed. In the Late Triassic, a depressional lake basin was formed within the Ordos Basin. Bivalve, crustacean and fish body fossils support a lake environment in a semi-arid to humid climate. Discoveries of horseshoe crab and diverse fish traces indicate a rich shallow lake epibenthic community.

Four Elegies

Four Elegies

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

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

Living fossils and conservation values

Horseshoe crabs (Limulus polyphemus) have been in decline in Long Island Sound, and recently there has been discussion of whether the state of Connecticut should stop issuing licenses for commercial harvesting. This paper argues that in spite of concerns about the living fossil concept, the fact that the horseshoe crabs are living fossils should count in favor of more stringent protection. The paper distinguishes four different views about the status of the living fossil concept: 1) eliminativism; 2) redefinition; 3) reframing; and 4) conceptual pluralism. Approaches 2–4 all treat the criteria associated with living fossils as picking out distinctive features of evolutionary history. Those distinctive features of evolutionary history link up with conservation values in several ways. More generally, drawing upon relevant work in environmental philosophy, it is argued that evolutionary history is relevant to aesthetic and environmental value. Moreover, eliminativists have trouble rendering intelligible a striking pattern in the recent scientific literature. Researchers undertaking conservation-relevant work frequently highlight the living fossil status of the taxa under study.

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

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

Risk of false conformity decisions in LAL gel-clot test due to false-negative results

Endotoxins are impurities capable of causing multiple deleterious effects to health. Therefore, the results of Limulus horseshoe crab amebocyte lysate (LAL) coagulation test in injectable drugs should be reliable. However, the reliability of qualitative results is still a challenge for analytical chemists and microbiologists. Thus, the objective of this work is to estimate the risk of false compliance/non-compliance decisions in LAL test based on the probability of true-negative and false-negative results through likelihood ratio value (LHR). For this, 11 injectable drugs were analyzed in two dilution schemes. A total of 1560 tests were performed using 41 different lots of LAL. The results from the confirmation of lysate sensitivity were used to obtain a logistic regression model, thus enabling to estimate the probability of negative results and consequently the LHR of the drug tests. LHR values were found to be between about 102 (114) and 105 (117,206), which corresponds to evidence of moderately strong to extremely strong negative results. Higher LHR values were obtained using lower dilution schemes. The use of logistic regression and calculation of LHR values can be useful to assess the reliability of qualitative results obtained by other chemical or microbiological methods.

Signs of Life – Hearts, Blood and our Breath

In their piece ‘Signs of Life - Hearts, Blood and our Breath: An artistic dialogue on embodiment and boundaries’, the authors and interdisciplinary artists Jane Prophet and Tobias Klein discuss three recent collaborative artworks made with Victor Leung and contextualise those works by referencing their individual art practices. Sensemaking with the heart and blood is central to these kinetic installation artworks: ‘Blood work 1.0’, ‘Blood work 2.0: Unruh’, and ‘Common Datum’. The trio made them by interweaving methods and approaches from arts-based inquiry and computer science within a feminist technoscience framework. Blood has historically been a powerful and often contested symbol in art, described by Anne Pollock, Professor of Global Health and Social Medicine, as ‘an object itself and an object within a network of objects’. This article considers the characteristics of blood and what it means to be human by triangulating between the chemical, physical and mechanical characteristics of blood. We focus on blood drawn from both human and nonhuman sources and the passage of air into the blood via the semi-permeable lung-heart connection. The artworks are inspired by the transfusion of blood and the use of organs donated by humans and extracted from nonhumans like horseshoe crabs. These biologically inspired, transparent kinetic sculptures depend on and transform the properties and qualities of blood in different species. The fluidity, coagulation and metal content of blood form the structures and movement of the sculptures. In addition, the artists incorporate shifting energies generated through visible mechanical and invisible magnetic forces to question-through-making the material properties and state change in blood flows.

Horseshoe crab trace fossils Arborichnus Romano et Meléndez, 1985 from the Bashkirian (Carboniferous) of the Donets Basin, Ukraine

This paper describes resting traces of horseshoe crabs Arborichnus repetitus Romano et Meléndez, 1985 from marine deposits of the Dyakove Group (Viséan–Bashkirian) and the Bashkirian coal-bearing rocks of the Mandrykyne and Mospyne formations in the Donets Basin, eastern Ukraine. The presence of Arborichnus repetitus indicates the widespread distribution of horseshoe crabs in the coastal paleobiocenoses of the Dyakove Paleobasin, located in the central part of the Donets Basin. The horseshoe crab resting traces Arborichnus repetitus from the Bashkirian strata of the Donets Basin are typical of deltaic and prodeltaic rocks, usually represented by sandstones and siltstones. Horseshoe crabs are the most likely trace-makers of Arborichnus Romano et Meléndez, 1985, but some other arthropods, e.g., aglaspidids, could also have produce these trace fossils. The discovery of the presumably horseshoe crab thoracetron on the same sandstone bedding plane as the traces Arborichnus indirectly confirms that xiphosurans were the trace-makers of these ichnofossils.

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.

Evaluation of Recombinant Cascade Reagent PyroSmart NextGen<sup>®</sup> and Limulus Amebocyte Lysate Equivalency in a Plate and Tube Reader for Bacterial Endotoxins Testing

PyroSmart NextGen® is a recombinant cascade reagent (rCR) for the detection and quantification of bacterial endotoxins developed using cloned genes derived from the Limulus polyphemus horseshoe crab genome. Requirements for use of this alternative reagent include analysis of analytical performance, method suitability, and test result equivalency to Limulus amoebocyte lysate (LAL) reagents used in the compendial Bacterial Endotoxins Test (BET). The plate reader evaluation has been expanded to address two long-standing user preferences, with the inclusion of the tube reader method increasing the sensitivity of endotoxin detection from 0.005 EU/mL to 0.001 EU/mL. The utilization of PyroSmart NextGen® with the two different instrument types also allows for a more comprehensive equivalency analysis. Furthermore, the comparison results demonstrate that PyroSmart NextGen® detects equivalent levels of autochthonous endotoxin in water samples when compared to LAL reagents. Overall, this study provides the first large-scale example of equivalency analysis utilizing a robust rCR and has verified that PyroSmart NextGen® meets the expectations for alternative reagents.