Horseshoe Crab Species Research Articles

Ontogenetic changes in dietary carbon sources and trophic position of two co-occurring horseshoe crab species in southwestern China

Ontogenetic changes in dietary carbon sources and trophic position of two co-occurring horseshoe crab species in southwestern China

Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products.

Recombinant Factor C (rFC) is non-animal-derived reagent used to detect bacterial endotoxins in pharmaceutical products. Despite the fact that the reagent was first commercially available nearly 15 years ago, the broad use of rFC in pharmaceutical industry has long been lagging, presumably due to historical single-source supplier concerns and the lack of inclusion in worldwide pharmacopeias. Commercial rFC reagents are now available from multiple manufacturers, thus single sourcing is no longer an issue. We report here the successful validation of several pharmaceutical products by an end-point florescence-based endotoxin method using the rFC reagent. The method is equivalent or superior to the compendia bacterial endotoxins test method. Based on the comparability data and extenuating circumstances, the incorporation of the end point fluorescence technique and rFC reagent in global compendia bacterial endotoxins test chapters is desired and warranted.LAY ABSTRACT: Public health has been protected for over 30 years with the use of a purified blood product of the horseshoe crab, limulus amebocyte lysate. More recently, this blood product can be produced in biotech manufacturing processes, which reduces potential impacts to the horseshoe crab and related species dependent upon the crab, for example, migrating shorebirds. The pharmaceutical industry has been slow to adopt the use of this reagent, Recombinant Factor C (rFC), for various reasons. We evaluated the use of rFC across many pharmaceutical products, and in other feasibility demonstration experiments, and found rFC to be a suitable alternative to the animal-derived limulus amebocyte lysate. Incorporation of rFC and its analytical method into national testing standards would provide an equivalent or better test while continuing to maintain patient safety for those who depend on medicines and while securing pharmaceutical supply chains. In addition, widespread use of this method would benefit existing animal conservation efforts.

Biodiversity and Distribution of Horseshoe Crabs in Northern Coast of Java and Southern Coast of Madura

Horseshoe crab is an important component of macro-benthos communities in the fine sand or mud substrate in coastal waters, both in the tropical and temperate region. This primitive animal consists of four species in the world, and three species can be found di Asian region, including Indonesia, namely Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda. Scientific information about species distribution of three Asian horseshoe crab in Indonesia is limited, also about morphometric characters. This study aims to determine the morphometric characters and species distribution of three Asian horseshoe crab in north coast of Java and south coast of Madura Island. This study was conducted on July-August 2016. The total number of three Asian horseshoe crab obtained in this study was 260 individuals, distributed along north coast of Java and south coast of Madura Island, respectively 176 individuals of C. rotundicauda, 35 individuals of T. tridentatus, and 49 individuals of T. gigas. Tachypleus gigas has the largest size and widest class interval among three Asian horseshoe crab species. Morphometric characters is differences among three Asian horseshoe crab species. Carapace width and telson length were not significantly different among sampling locations only in T. tridentatus.

Opsin Repertoire and Expression Patterns in Horseshoe Crabs: Evidence from the Genome of Limulus polyphemus (Arthropoda: Chelicerata).

Horseshoe crabs are xiphosuran chelicerates, the sister group to arachnids. As such, they are important for understanding the most recent common ancestor of Euchelicerata and the evolution and diversification of Arthropoda. Limulus polyphemus is the most investigated of the four extant species of horseshoe crabs, and the structure and function of its visual system have long been a major focus of studies critical for understanding the evolution of visual systems in arthropods. Likewise, studies of genes encoding Limulus opsins, the protein component of the visual pigments, are critical for understanding opsin evolution and diversification among chelicerates, where knowledge of opsins is limited, and more broadly among arthropods. In the present study, we sequenced and assembled a high quality nuclear genomic sequence of L. polyphemus and used these data to annotate the full repertoire of Limulus opsins. We conducted a detailed phylogenetic analysis of Limulus opsins, including using gene structure and synteny information to identify relationships among different opsin classes. We used our phylogeny to identify significant genomic events that shaped opsin evolution and therefore the visual system of Limulus. We also describe the tissue expression patterns of the 18 opsins identified and show that transcripts encoding a number, including a peropsin, are present throughout the central nervous system. In addition to significantly extending our understanding of photosensitivity in Limulus and providing critical insight into the genomic evolution of horseshoe crab opsins, this work provides a valuable genomic resource for addressing myriad questions related to xiphosuran physiology and arthropod evolution.

Co-occurrence of juvenile horseshoe crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda in an estuarine bay, southwestern China

Spawning and nursery habitats are critical in the conservation of horseshoe crabs. We examined the abundance and distribution of juveniles of 2 species of horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda, in a mangrove-vegetated estuary (Pearl Bay, Guangxi, China). Intertidal sampling and measurement of environmental variables were carried out in October 2014 at 4 sites spanning ~6.3 km from the inner to the outer bay. Both species were found at the 2 innermost mangrove sites but not at the outer 2 sites. Densities were higher than recorded in previous studies, 0.24 ind. m �2 for C. rotundicauda and 0.05 ind. m �2 for T. tridentatus. Although only 12 of the latter species were found, they appeared to be more abundant in more sea- ward, energetic environments in poorly sorted sediments, whereas C. rotundicauda occurred in sediments with higher silt-clay and organic carbon content, and aggregated at the mangrove forest edges. These small-scale distribution differences are consonant with the species' life histories, since only T. tridentatus migrates to the sea while C. rotundicaudata spends its entire life within or near mangrove habitats. International and national conservation strategies for these species should include further efforts to inventory critical habitats, such as Pearl Bay, in southeast Asia.

Ancestral whole-genome duplication in the marine chelicerate horseshoe crabs.

Whole-genome duplication (WGD) results in new genomic resources that can be exploited by evolution for rewiring genetic regulatory networks in organisms. In metazoans, WGD occurred before the last common ancestor of vertebrates, and has been postulated as a major evolutionary force that contributed to their speciation and diversification of morphological structures. Here, we have sequenced genomes from three of the four extant species of horseshoe crabs-Carcinoscorpius rotundicauda, Limulus polyphemus and Tachypleus tridentatus. Phylogenetic and sequence analyses of their Hox and other homeobox genes, which encode crucial transcription factors and have been used as indicators of WGD in animals, strongly suggests that WGD happened before the last common ancestor of these marine chelicerates >135 million years ago. Signatures of subfunctionalisation of paralogues of Hox genes are revealed in the appendages of two species of horseshoe crabs. Further, residual homeobox pseudogenes are observed in the three lineages. The existence of WGD in the horseshoe crabs, noted for relative morphological stasis over geological time, suggests that genomic diversity need not always be reflected phenotypically, in contrast to the suggested situation in vertebrates. This study provides evidence of ancient WGD in the ecdysozoan lineage, and reveals new opportunities for studying genomic and regulatory evolution after WGD in the Metazoa.

Chirotherium trackways and feeding traces on seismic-influenced carbonate intertidals of the Middle Triassic of Central Europe: global food chain reactions onto horseshoe crab reproduction mud flat beaches of the Germanic Basin

Newly excavated Middle Triassic reptile Chirotherium, Rhynchosauroides and horseshoe crab Kouphichnium imprints from the uppermost Pelsonian megatracksite Bernburg, Germany (Central Europe) represent different behavioural trackways. The Chirotherium/Rhynchosauroides/Kouphichnium track assemblage of the lower intertidal carbonate flats is dominated by well-known horseshoe crab tracks of various subaquatic benthic movements which were all left on mud-cracked biolaminites in reproduction coastal zones. Most abundant reptile Rhynchosauroides trackways (50 % of reptile tracks) are oriented more beach-parallel (to Rhenish Massif Island) as a result of food searching along the intertidal seismic-influenced coast (16 slickensided biolaminate layers within 2 m biolaminates due to Alpine fold belt and Germanic intracratonic Basin tectonics), most probably for feeding on high abundant horseshoe crab eggs resulting from mating events in the lower intertidal. Large basal archosaur predators (?Ticinosuchus) produced large Chirotherium trackways (25 % of reptile tracks) which are represented in the new material with exceptional unusual trackways (slipping and side steps), but in directions to and from the beach to the Rhenish Massif main land, indicating predation on small reptiles. Finally, medium-sized Chirotherium trackways of subadult animals are present (25 % of reptile tracks). One trackway has a unique “feeding place” area, indicating the unexpected either predation, playing or feeding of a basal archosaur on a horseshoe crab. The horseshoe crab migrations from the North Tethys onto the extended mating intertidal mud flat beaches of the Germanic Basin, and recently known even into Chinese lagoons migrating limulid populations of different horseshoe crab species must have caused a global and seasonal food chain reaction, whereas Macrocnemus reptiles (Rhynchosauroides trackmaker) might have fed only on Millions of their eggs.

A collection of horseshoe crabs (Chelicerata: Xiphosura) in the National Museum, Prague (Czech Republic) and a review of their immunological importance

The zoological collection of the National Museum, Prague (NMP) contains spirit (juvenile) as well as dry (mostly adult) specimens of horseshoe crabs (Xiphosura). Living horseshoe crabs are of immunological importance due to clotting agents present in their hemolymph. Here we summarize basic data about the mechanism of the immune system of these marine animals and its use in practice – the Limulus Amebocyte Lysate test – including the laboratory assays and handling with the animals. In the NMP collection, 82 specimens (16 dry and 66 spirit) of all four currently recognised living species of horseshoe crabs are present. They were collected in Indonesia, USA and Vietnam in 1872–1998; Limulus polyphemus from the USA is the most numerous species in the NMP. The collection contains no type specimens but three historical mounted ontogenetic series are present. The largest part of the horseshoe crab collection is 55 spirit specimens from the collection of Vaclav Fric (1839–1916) whose preparations were intended mostly for educational purposes.

Levels and patterns of genetic variation in an Asian horseshoe crab species, Tachypleus gigas Müller, from the Malay Peninsula

Horseshoe crabs are one of the oldest living organisms that still exist today. Given the overexploitation of horseshoe crabs around the world, and the lack of information on their populations, it is crucial that genetic variation studies are done to assess their levels and patterns of genetic variation for continuous monitoring and management of their populations. Here, five populations of the Asian horseshoe crab Tachypleus gigas sampled from along the coasts of the Malay Peninsula were studied using simple-sequence repeat (SSR) and inter-simple-sequence repeat (ISSR) markers. Different results were obtained using the two different types of markers in terms of the levels of genetic variation estimated, but both concurred that most of the genetic variations were distributed at the individual level rather than among populations of the species. Inbreeding was also observed using the SSR data, although the presence of null alleles could have influenced the estimation. Finally, both marker types revealed that T. gigas could have been subjected to the land barrier effect of the Malay Peninsula that causes populations from the Indian Ocean to be genetically differentiated from the populations from the South China Sea.

The Malay Peninsula as a barrier to gene flow in an Asian horseshoe crab species, Carcinoscorpius rotundicauda Latreille

Horseshoe crabs are marine arthropods that are amongst the oldest living creatures that still exist today. Among the four extant species of horseshoe crabs, Carcinoscorpius rotundicauda differs from the other species by having poisonous eggs and lays its eggs in sandy-mud areas near river mouths. With the rapid development of coastal areas worldwide, C. rotundicauda habitats are decreasing. Until now, however, there has not been any study on the species' genetic variation. Simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers were employed to study the genetic variation in five C. rotundicauda populations from the east and west coasts of the Malay Peninsula. Both markers showed differing levels of genetic variation, but concurred on the pattern of genetic structuring among populations of the species. This includes showing that little, although significant, genetic differentiation is present among populations, suggesting a low rate of gene flow among populations. The results also suggested that C. rotundicauda may be subjected to the land barrier effect of the Malay Peninsula, whereby gene flow is limited between populations occurring on both sides of the peninsula, increasing their genetic differentiation through time.

The horseshoe crab of the genus Limulus: living fossil or stabilomorph?

A new horseshoe crab species, Limulus darwini, is described from the uppermost Jurassic (ca. 148 Ma) near-shore sediments of the Kcynia Formation, central Poland. The only extant species Limulus polyphemus (Linnaeus) inhabits brackish-marine, shallow water environments of the east coast of the United States. Here it is shown that there are no important morphological differences between the Kcynia Formation specimens and extant juvenile representatives of the genus Limulus. The palaeoecological setting inhabited by the new species and the trophic relationships of extant horseshoe crabs are discussed in an attempt to determine the potential range of food items ingested by these Mesozoic xiphosurans. In this paper we propose the adoption of a new term stabilomorphism, this being: an effect of a specific formula of adaptative strategy among organisms whose taxonomic status does not exceed genus-level. A high effectiveness of adaptation significantly reduces the need for differentiated phenotypic variants in response to environmental changes and provides for long-term evolutionary success.

Oxygen consumption in trilobite larvae of the mangrove horseshoe crab (Carcinoscorpius rotundicauda; Latreille, 1802): effect of temperature, salinity, pH, and light–dark cycle

Carcinoscorpius rotundicauda is one of the four extant species of horseshoe crab, occurring only in Asia: around India, Indonesia, Malaysia, Philippines, Singapore, Thailand and Hong Kong. Virtually nothing is known about the physiology of this Asian species. In the present study, the respiratory physiology in terms of oxygen consumption rate of the trilobite larvae of C. rotundicauda was evaluated under laboratory conditions. The trilobite larvae were exposed to different levels of temperature (10, 20, 30 and 40 °C), salinity (10, 20, 30 and 40 ppt), pH (5, 6, 7, 8 and 9) and dark–light conditions for a period of 12 h. Effect of temperature on oxygen consumption in trilobite larvae was not so significant among all the temperatures tested. However, both 10 and 40 °C temperature showed comparatively high rate of oxygen consumption at the initial 4 h of experiment. More or less consistent oxygen consumption pattern observed at 20 and 30 °C temperature suggested 20–30 °C as the best temperature range for rearing trilobite larvae. Trilobite larvae revealed enormous tolerance to temperature and indicated that the temperature is not critical in terms of respiration. The trend in oxygen consumption was more or less uniform at all salinities tested indicating the insignificant influence of salinity on respiration. Though not significant, the oxygen consumption trend was more consistent at 20 ppt as compared to other salinities and thus could be considered as the most suitable salinity for rearing trilobite larvae. There was a positive relationship between oxygen consumption rate and the various seawater water pH tested with trilobite larvae. Maximum oxygen consumption was recorded between pH 7 and 9 at the initial hours of the experiment and the overall trend suggests that the larvae preferred slightly acidic conditions. Although the larvae showed higher oxygen consumption under light, in the overall analysis of the data the light–dark condition did not have a statistically significant influence. However, more consistent oxygen consumption pattern and swimming activity in the dark condition as compared to light condition during the 12 h of experiment revealed that the larvae have active nocturnal behaviour.

Digestible dietary protein and energy requirements of juvenile Asian horseshoe crabs,Tachypleus tridentatusandCarcinoscorpius rotundicauda

A 84-day feeding experiment of two juvenile horseshoe crab species, including nine formulated diets with three digestible protein (DP) levels (36%, 40% and 44%) and three digestible energy (DE) levels (14, 16 and 18 kJ g−1) versus the control, fed on frozen brine shrimp (Artemia salina), was conducted. The results showed that the survival rate of Tachypleus tridentatus ranged from 67% to 100%, while that of Carcinoscorpius rotundicauda varied from 44% to 100%. The results also indicated that the formulated diet containing 40% DP with 14 kJ g−1 DE was the best, in terms of growth and feed utilization, for the two juvenile horseshoe crab species. Final body weight (FBW), thermal-unit growth coefficient (TGC), feed efficiency (FE), nitrogen retention efficiency (NRE) and energy retention efficiency (ERE) of T. tridentatus fed 40% DP and 14 kJ g−1 DE were significantly higher than that of T. tridentatus fed the control diet. However, for C. rotundicauda, only significantly higher FE, NRE and ERE were found in juveniles fed 40% DP and 14 kJ g−1 DE than the control diet. The present findings revealed that the optimum DP/DE ratio for the two species was 28 g protein MJ−1.

Remarkable preservation of a new genus and species of limuline horseshoe crab from the Cretaceous of Texas, USA

Abstract: A single specimen, part and counterpart of a carapace, of a horseshoe crab from the Lower Cretaceous (Albian) Glen Rose Formation in north‐central Texas, forms the basis for the definition of a new genus and species, Crenatolimulus paluxyensis. The discovery represents only the fifth limuline known from the Cretaceous. Its preservational style is remarkable in that the carapace exterior is faithfully replicated by a massive overgrowth of serpulid worms.

Intraspecific shape variation in horseshoe crabs: The importance of sexual and natural selection for local adaptation

A morphometric analysis of the body shape of three species of horseshoe crabs was undertaken in order to infer the importance of natural and sexual selection. It was expected that natural selection would be most intense, leading to highest regional differentiation, in the American species Limulus polyphemus, which has the largest climatic differences between different populations. Local adaptation driven by sexual selection was expected in males but not females because horseshoe crab mating behaviour leads to competition between males, but not between females. Three hundred fifty-nine horseshoe crabs from nine populations, representing three species, were analyzed using a digitizer to position sixty morphometric landmarks in a three-dimensional space. Discriminant analysis revealed strong regional structuring within a species, which suggests strong philopatry, and showed the existence of geographically-based intraspecific variation. An admixture analysis showed regional intraspecific differentiation for males and females of L. polyphemus and males of the Asian horseshoe crab Carcinoscorpius rotundicauda, but not for females of C. rotundicauda and another Asian horseshoe crab, Tachypleus gigas. Differences in shape variation between sexes were tested with F-tests, which showed lower intrapopulation morphometric variation in males than females. These results indicate a lower degree of local adaptation on body shape in C. rotundicauda and T. gigas than in L. polyphemus and a lower degree of local adaptation in females than in males.

Evolution of the thioester-containing proteins (TEPs) of the arthropoda, revealed by molecular cloning of TEP genes from a spider, Hasarius adansoni

The thioester-containing protein (TEP) family of genes, found in most Eumetazoan genomes, is classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily and the C3 subfamily. Many A2M subfamily members, including insect TEP (iTEP), have been reported from the Arthropoda, whereas the C3 subfamily members have been reported only from two horseshoe crab species thus far. To elucidate the evolution of these genes among the Arthropoda, TEP genes were isolated from a spider, Hasarius adansoni (Chelicerata), by reverse transcription polymerase chain reaction (RT–PCR) amplification using universal degenerate primers specific for the thioester region. Four different TEP genes were identified. Phylogenetic analysis using the entire amino acid sequences of these and various other TEP sequences from the Eumetazoa indicated that two of the spider genes are type C3 ( HaadC3-1 and HaadC3-2), one is type A2M ( HaadA2M) and the other is closely related to iTEP ( HaadiTEP). These results suggest that the common ancestor of the Arthropoda possessed at least three TEP genes, C3, A2M and iTEP and that they were lost differentially in the Crustacean and Hexapodan lineages.

Molecular Phylogeny of Horseshoe Crab

An effort to assess the utility of 650 bp Cytochrome C oxidase subunit I (DNA barcode) gene in delineating the members horseshoe crabs (Family: xiphosura) with closely related sister taxa was made. A total of 33 sequences were extracted from National Center for Biotechnological Information (NCBI) which include horseshoe crabs (Class: Merostomata), beetles (Class: Insecta), common crabs (Class: Melacostraca) and scorpion (Class: Arachnida) sequences. Constructed phylogram through comprehensive dry lab methodology using advanced software predictive tools showed that beetles are closely related with horseshoe crabs than common crabs (Class: Melacostraca). It was interesting to note that terrestrial Scorpion (Class: Arachnida) were distantly related to horseshoe crabs (Class: Merostomata). Phylogram and observed Genetic Distance (GD) data were also revealed that Limulus polyphemus was distantly related to all the other horseshoe crab species. Tachypleus gigas was closely related with Carcinoscorpius rotundicauda than with Tachypleus tridentatus. The observed mean Genetic Distance (GD) value was higher in 3rd codon position in all the selected group of organisms. Among the horseshoe crabs high GC content was observed in L. polyphemus (38.32%) and lowest was observed in T. tridentatus (32.35%). We conclude that COI sequencing (barcoding) could be used in identifying and delineating evolutionary relatedness with closely related species

Effect of starvation on the energy budget of two Asian horseshoe crab species: Tachypleus tridentatus and Carcinoscorpius rotundicauda (Chelicerata: Xiphosura)

Energy budget is one of the most studied parameters in aquatic animals under environmental challenge. To examine how prolonged starvation would affect their energy budget, respiration rate (RR), ammonia excretion rate (ER), oxygen consumption to ammonia–nitrogen excretion (O:N) ratio and scope for growth (SfG) representing the balance between energy intake and metabolic output, two Asian horseshoe crab species, Tachypleus tridentatus and Carcinoscorpius rotundicauda, were investigated in two feeding regimes (fed and starved) over a period of 7 weeks. No significant effects of species and time course, as well as their interaction, on absorption efficiency were observed in the fed treatments. For both species, RR and ER of the starved treatments significantly decreased, while their O:N ratio significantly increased during the experiment. However, such values for the fed treatments remained relatively stable over the study period. A rapid reduction in SfG was only apparent in the first week of the starved treatments for both species; thereafter, their SfG remained relatively constant. In the fed treatments, SfG of T. tridentatus was significantly lower than that of C. rotundicauda throughout the experiment. In general, C. rotundicauda showed a greater decrease in SfG under starvation than T. tridentatus, suggesting that they may have a more competitive life-history strategy for adjusting to poor nutritional conditions.

Reproductive behavior in horseshoe crabs: Does density matter?

Abstract While the four species of horseshoe crabs share many common reproductive traits with respect to their reproductive systems, they do differ with respect to their mating behavior (monogamy vs. polygynandry). Past research has attributed these differences to a number of factors including: spawning densities, operational sex ratios (OSR's), male condition (or age), environmental and/or genetic factors, or a combination thereof. Mating behaviors in the three Asian horseshoe crab species (Tachypleus gigas, T. tridentatus, and Carcinoscorpius rotundicauda) with low spawning densities and 1:1 operational sex ratios are typically monogamous. In Limulus polyphemus, mating behavior is more variable ranging from monogamy to polygynandry. Here we provide evidence, through a long term behavioral study, that variation in mating behavior is influenced by population density in L. polyphemus. Our study population on two beaches in Connecticut (Long Island Sound) have a spawning density 400 times less than that found in Delaware Bay (0.002 females/m2 vs. 0.8 females/m2) but similar operational sex ratios. Between 90%-95% of all spawning females in CT were paired with only one male, thus exhibiting monogamous behavior. In contrast, between 30 and 60% of spawning females in Delaware Bay have more than one mate and produce clutches of eggs with multiple paternities. Male condition played no role in mating behavior in CT populations. We also observed that on average 18% of the females on the spawning beaches are single. These results suggest that population density is an important condition that determines mating behavior. Also, low population density may lead to decreased mate finding ability and lost opportunities for spawning.

Effect of prolonged starvation on body weight and blood-chemistry in two horseshoe crab species: Tachypleus tridentatus and Carcinoscorpius rotundicauda (Chelicerata: Xiphosura)

To determine the effect of prolonged starvation on body weight and blood-chemistry of two Asian horseshoe crab species, Tachypleus tridentatus and Carcinoscorpius rotundicauda, the animals were stocked into indoor recirculation systems under two food treatments (fed and starved) for seven weeks. Seven blood-chemistry parameters: alkaline phosphatase, calcium, cholesterol, glucose, lipase, triglyceride and total protein of plasma, were measured. During the experimental period, no significant differences of body weight were detected between the starved group and fed group in the two species. Plasma alkaline phosphatase increased slightly and then decreased. Plasma calcium remained fairly constant during the experimental period. A continuous decrease of plasma glucose in the starved treatment was observed. Additionally, cholesterol, lipase and triglyceride in the plasma increased significantly at later times, while plasma total protein remained fairly constant during the experiment. Significant interactive effects between feeding regime and starved time on some parameters were also evident in the two species. The results suggested that, for adult horseshoe crabs, starvation may stimulate a “hunger response”, which in turn might lead to consumption of some nutrient reserves in the animals. By consuming such nutrient reserves, adult horseshoe crabs can withstand prolonged starvation. The present findings contribute to the understanding of the blood biochemical aspect of the biology of T. tridentatus and C. rotundicauda, when these animals encounter starvation in the wild.