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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]

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]

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]

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]

The Delaware Bay region is the epicenter of horseshoe crab, Limulus polyphemus, activity, and despite the ecological and commercial importance of this species, few studies have examined the long-term movements of horseshoe crabs in this area and the amount of mixing that takes place between smaller coastal embayments within the region and the Delaware Bay proper, factors that are critical to effective management. To better understand these factors, 5568 crabs were tagged in the Delaware Inland Bays as part of the U.S. Fish and Wildlife Service’s (USFWS) Cooperative Horseshoe Crab Tagging Program in 2002-2016. A high re-sight rate of 20.1% (1123 crabs) was reported to the USFWS. Re-sights suggest that the Delaware Bay population is distributed between coastal New Jersey (south of Barnegat Bay) and coastal Virginia (north of Chincoteague Inlet). There were 90 re-sights in the Inland Bays and 148 re-sights in Delaware Bay, with 320 days or more between tagging and re-sight, showing that substantial interchange between successive spawning seasons occurs. Distance analyses demonstrated that crabs can move between the Inland Bays and other Delaware Bay region waterbodies within a single year. The findings of this study support the current management strategy of splitting the harvest of Delaware Bay crabs between New Jersey, Delaware, Maryland, and Virginia and also demonstrate that the waterbodies within the Delaware Bay region are highly connected. This connectivity supports protecting spawning habitat within the smaller embayments of the Delaware Bay region and including spawning surveys from these systems in future stock assessments. [ABSTRACT FROM AUTHOR]

Restoration of horseshoe crab spawning habitats through beach nourishment may be considered as a potential strategy to enhance reproductive success in areas where estuarine beaches have been lost to coastal erosion and development. The US Army Corps of Engineers performed a beach nourishment project at Plumb Beach (Jamaica Bay, Brooklyn, NY) in 2012 to stabilize the shoreline. While the addition of sand was done to protect infrastructure, it created an opportunity to examine the responses of American horseshoe crabs (Limulus polyphemus) to beach nourishment using a BACI (before-after control impact) design. During Spring 2012, before beach nourishment, horseshoe crabs made minimal use of the highly degraded western section of Plumb Beach in comparison to a nearby reference site, as quantified by numbers of spawning adults at high tide and densities of horseshoe crab eggs in core samples. In the first post-nourishment field season (Spring 2013), there was no detectable increase in horseshoe crab spawning activity on the newly restored beach. In 2014 and 2015, the density of spawning females began to increase at the nourished beach, although their numbers and especially the density of horseshoe crab eggs remained much lower than at the reference site. Three years after beach nourishment, differences in sediments texture (mean grain diameter, percent gravel, sorting, skewness, and hardness) were still evident between the nourishment and reference sites. Our results suggest that (1) at this site, beach nourishment appeared to bring about only slow increases in horseshoe crab spawning density after several seasons and (2) subtle differences in beach geomorphology over relatively short distances can be detected by horseshoe crabs and may underlie their selection of specific nesting sites. [ABSTRACT FROM AUTHOR]

Horseshoe crabs have persisted for more than 200 million years, and fossil forms date to 450 million years ago. The American horseshoe crab ( Limulus polyphemus), one of four extant horseshoe crab species, is found along the Atlantic coastline of North America ranging from Alabama to Maine, USA with another distinct population on the coasts of Campeche, Yucatán and Quintana Roo in the Yucatán Peninsula, México. Although the American horseshoe crab tolerates broad environmental conditions, exploitation and habitat loss threaten the species. We assessed the conservation status of the American horseshoe crab by comprehensively reviewing available scientific information on its range, life history, genetic structure, population trends and analyses, major threats, and conservation. We structured the status assessment by six genetically-informed regions and accounted for sub-regional differences in environmental conditions, threats, and management. The transnational regions are Gulf of Maine (USA), Mid-Atlantic (USA), Southeast (USA), Florida Atlantic (USA), Northeast Gulf of México (USA), and Yucatán Peninsula (México). Our conclusion is that the American horseshoe crab species is vulnerable to local extirpation and that the degree and extent of risk vary among and within the regions. The risk is elevated in the Gulf of Maine region due to limited and fragmented habitat. The populations of horseshoe crabs in the Mid-Atlantic region are stable in the Delaware Bay area, and regulatory controls are in place, but the risk is elevated in the New England area as evidenced by continuing declines understood to be caused by over-harvest. The populations of horseshoe crabs in the Southeast region are stable or increasing. The populations of horseshoe crabs in the Florida Atlantic region show mixed trends among areas, and continuing population reductions at the embayment level have poorly understood causes. Within the Northeast Gulf of Mexico, causes of population trends are poorly understood and currently there is no active management of horseshoe crabs. Horseshoe crabs within México have conservation protection based on limited and fragmented habitat and geographic isolation from other regions, but elevated risk applies to the horseshoe crabs in the Yucatán Peninsula region until sufficient data can confirm population stability. Future species status throughout its range will depend on the effectiveness of conservation to mitigate habitat loss and manage for sustainable harvest among and within regions. [ABSTRACT FROM AUTHOR]

Populations of a single species may vary substantially in sexually selected traits. However, the majority of studies investigating such variation across populations have focused on traits important for pre-copulatory reproductive success, such as male ornamentation or song. Relatively fewer studies have investigated whether traits important in post-copulatory sexual selection vary across populations. In this study, we examine how sperm and ejaculate quantity and quality in the American horseshoe crab, Limulus polyphemus differ at six sites, from Maine to Florida. Horseshoe crabs from these sites differ in many respects, including average body size, age, and the risk and intensity of sperm competition as measured by the operational sex ratio. We find that sperm and ejaculate quantity and quality significantly differ across this range and that this variation persists after taking body size and age into account. Sperm traits also do not follow strictly linear latitudinal clines. Finally, we find that the site-specific operational sex ratio correlates with changes in sperm and ejaculate quantity. This latter result may indicate that the same processes that lead to variation in post-copulatory sexually selected traits across related species may also promote trait divergence across populations of the same species. Significance statement: This study describes variation in sperm and ejaculate traits from horseshoe crabs collected at six sites from Maine to Florida. While geographic variation has been described for many traits, relatively few studies have demonstrated that sperm and ejaculate traits follow similar patterns. The variation we found does not follow simple latitudinal patterns, suggesting that other forces may be driving sperm and ejaculate trait divergence across sites. We also have data that suggest sexual selection may play a role in driving sperm trait variation, but we caution that additional sites need to be sampled. [ABSTRACT FROM AUTHOR]

The Great Bay Estuary, New Hampshire, USA is near the northern distribution limit of the American horseshoe crab ( Limulus polyphemus). This estuary has few ideal beaches for spawning, yet it supports a modest population of horseshoe crabs. There is no organized monitoring program in the Great Bay Estuary, so it is unclear when and where spawning occurs. In this 2-year study (May through June, 2012 and 2013), >5,000 adult horseshoe crabs were counted at four sites in the estuary. The greatest densities of horseshoe crabs were observed at Great Bay sites in the upper, warmer reaches of the estuary. Peaks of spawning activity were not strongly correlated with the times of the new or full moons, and similar numbers of horseshoe crabs were observed mating during daytime and nighttime high tides. While many environmental factors are likely to influence the temporal and spatial patterns of spawning in this estuary, temperature appears to have the most profound impact. [ABSTRACT FROM AUTHOR]

In systems with male alternative reproductive tactics, discrete behavioral patterns often lead to unequal levels of sperm competition across tactics. In many of these systems, the disadvantaged tactic shows adaptations to sperm traits that increase the competitive ability of its sperm. However, many studies have found that not all sperm traits differ across tactics, and in some systems no sperm trait adaptations are found. Why some systems and some sperm traits display tactic-specific differences and others do not is not always clear. Here, we investigate three sperm traits-ejaculate size, sperm concentration, and sperm velocity-in the American horseshoe crab, Limulus polyphemus, a system where males have condition-dependent reproductive tactics and females influence the amount of sperm competition males face during spawning. Contrary to our predictions that the disadvantaged tactic would show sperm trait adaptations, we found that horseshoe crabs of the tactic that faces the least amount of sperm competition had more concentrated sperm than the males of the other tactics. We propose a number of hypotheses to explain this result and suggest that future studies of alternative reproductive tactics might benefit from considering the role of females in influencing tactic-specific sperm trait adaptations. [ABSTRACT FROM AUTHOR]

Construction of bulkheads is a common response to erosion of estuarine shorelines. Bulkheads are usually built incrementally, resulting in wider sandy beaches remaining as enclaves between bulkhead segments. This paper measures the characteristics of bulkheads and enclaves and evaluates (1) whether horseshoe crabs ( Limulus polyphemus) utilize enclaves for spawning when they provide partial sheltering during periods of high wave energies; and (2) whether eggs become trapped in the enclaves and are available to shorebirds when the beaches in front of bulkheads are inundated. The characteristics of bulkheads and beach enclaves were identified in five developed reaches in Delaware Bay, USA. Counts of horseshoe crabs were made in enclaves in two of these reaches during times of high wave energies and compared to counts in nearby unarmored segments. Egg tracer and trapping experiments were conducted at one of the enclaves to assess egg movement. Results indicate that the percent of bulkheads intersecting the beach below mid-foreshore varies from 10 to 50 %. Spawning densities were greater in enclaves than on longer unarmored segments on some days. Enclaves serve as a sink for eggs moving along the base of the bulkheads. Most birds feeding on horseshoe crab eggs preferred sites outside the enclaves and bulkhead segments. Any advantage of bulkheads creating enclaves and sinks for eggs moving alongshore is likely overridden by their disruption to natural process and habitats, but having unarmored enclaves between bulkhead segments may be preferable to one continuous bulkhead, based on environmental benefits. [ABSTRACT FROM AUTHOR]

The health of horseshoe crab ( Limulus polyphemus) eggs is important not only to maintain horseshoe crab populations, but because they are a resource for higher trophic levels, such as fish and shorebirds. We examined the concentrations of arsenic, cadmium, chromium, lead, manganese, mercury, and selenium in the eggs of horseshoe crabs from Delaware Bay (between New Jersey and Delaware, USA) in 1993, 1994, 1995, 1999, 2000, and 2012 to determine if there were significant temporal changes and if levels appear to pose a health risk to the crabs themselves, or to predators that consume them. All metal levels declined in horseshoe crab eggs between 1994 and 2012, although the declines were much less consistent for lead and chromium than that for mercury and cadmium. Levels of contaminants found in these eggs are well below those known to cause adverse effects in the crabs themselves or to organisms that consume them, such as migrating shorebirds. [ABSTRACT FROM AUTHOR]

Populations of the American horseshoe crab ( Limulus polyphemus) differ in broad areas of their biology. We observed a non-harvested, marked Florida Gulf coast population during their spring spawning (March-May) in 11 years across a 17-year period (1992-2009). Long-term changes occurred in the number of spawning pairs: the population was stable from 1992 to 2000 but increased markedly after 2000. Short-term variation in numbers of spawning pairs, unpaired females, unpaired males, and operational sex ratios was explained by changes over the season and during each week of spring tides and by differences in actual (not predicted) maximum high tide height. Wind direction strongly affected tidal inundation and the number of spawning horseshoe crabs. Tagging individuals revealed that females returned to the nesting beach less often than males and most females were re-sighted only within 1 week of spring tides. No animals were seen across more than 6 years. Implications for management are discussed. [ABSTRACT FROM AUTHOR]

[H]-fructose and [H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species. [ABSTRACT FROM AUTHOR]

The spring spawning by the American horseshoe crab ( Limulus polyphemus L.) results in temporally and spatially discrete inputs of eggs onto sandy beaches in Delaware Bay, USA. We tested the hypothesis that seasonal patterns of sediment organic carbon on Delaware Bay beaches is linked to this pulsed input of horseshoe crab eggs. At a location with minimal horseshoe crab spawning activity (Higbee Beach), there was little seasonal variation in sediment organic carbon, no distinction between organic carbon levels as a function of shoreline position or sediment depth, and no significant correlation between the abundance of crab eggs and percent organic carbon. Conversely, at a prime horseshoe crab spawning habitat (North Beach), organic carbon levels were seasonally pulsed and were correlated with egg abundance. Moreover, the strongest evidence of seasonality was seen at the middle foreshore location at the 15-20 cm depth, consistent with the highest input of horseshoe crab eggs. Although some of the organic carbon contributed by horseshoe crab eggs in May-June leaves the beach in the form of hatched larvae later in the year, there is a net input of organic carbon to the system in the form of unfertilized and/or dead eggs, egg membranes, and embryonic molts. We suggest that the inputs of eggs from horseshoe crabs and other beach spawning animals, such as grunion and capelin, make significant contributions to the energy budget of sandy beaches. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) was detected by HPLC and LC-MS/MS in specimens of the horseshoe crab Carcinoscorpius rotundicauda collected from Tan Hai village, Vung Tau province, Vietnam, where cases of poisoning due to the consumption of horseshoe crab have frequently been reported. The soft tissue from 10 out of the 12 specimens analyzed showed considerable levels of TTX, making them unsafe for human consumption (81.2 ± 50.3 MU/g). Paralytic shellfish poisoning (PSP) toxins were also detected in all of the specimens, but the levels were low. These findings show that the frequency of occurrence of C. rotundicauda specimens with a high level of TTX is significantly high, and that the TTX is responsible for the food poisonings caused by the consumption of C. rotundicauda in this area. [ABSTRACT FROM AUTHOR]

Aim: To investigate the role of intercellular potassium in tachyplesin-induced HL-60 cells apoptosis. Methods: The concentration of intercellular potassium, cell volume and mitochondrial membrane potential were examined by flow cytometry. Results: The concentration of intercellular potassium reduced in a time-dependent manner in tachyplesin-treated HL-60 cells. In addition, the loss of mitochondrial membrane potential was tightly coupled with the shrinkage of cell volume. Different caspase inhibitors protected against DNA degradation but did not prevent the loss of HL-60 cell viability induced by tachyplesin. Ba2+, which was a kind of blocker of volume-regulatory K+ channels, increased the viability of tachyplesin-treated HL-60 cells and maintained mitochondrial membrane potential and cell volume. Conclusion: Efflux of K+ was an important reason for apoptosis in tachyplesin-treated HL-60 cells. Efflux of K+ affected the viability of tachyplesin-treated HL-60 cells independent of the process of caspase activation. [ABSTRACT FROM AUTHOR]

In the horseshoe crab mating system, mated pairs are frequently accompanied by unattached satellite males as they spawn on intertidal beaches. Previous studies have shown that males locate females visually using their lateral (compound) eyes, and that attached (mated) males generally have less heavily worn or damaged carapaces than unattached males. The purpose of this study was to investigate the influences of lateral eye condition and clasper abnormalities on male mating tactics. Sexually mature males had two kinds of eye damage: deterioration caused by disease, and overgrowth by sessile invertebrates, such as bryozoans, mussels, and tube-building polychaetes. The lateral eyes of attached males had significantly less decay than unattached males. On the other hand, coverage of the lateral eyes by encrusting invertebrates was more extensive among attached than unattached males. Although overgrowth did not appear to impair a male’s ability to pair with a female as severely as eye decay, it is conceivable that amplexus may have occurred before epibiont coverage was sufficient to obscure vision. Male crabs that were experimentally “blindfolded” by painting their lateral eyes with black nail polish were less likely to reattach to a female than controls. Appendage injuries were more frequent among unattached males than among attached males; in particular, 6.4% of unattached males but 0.0% of attached males had damaged claspers (the modified first legs required for amplexus). Unattached males in the population were “older,” as judged by the degree of carapace wear, than attached males. Severe visual impairment and/or clasper damage may explain the reduced pairing success of older male horseshoe crabs, and underlie their choice of the alternative satellite male mating tactics. [ABSTRACT FROM AUTHOR]

This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amerbocyte morphology was least altered from in vivo form in Grace’s Modified Insect Medium, with no observed degranulation of cells, as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell culture media tested. Grace’s Modified Insect Medium sustained viability of 77.2±5.1% (mean ± standard deviation) of amebocytes, followed distantly by Grace’s Insect Medium with 35.1±8.7% amebocyte viability. Results indicate that Grace’s Modified Insect Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization for Limulus amebocyte requirements. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine some general aspects of feeding in Tachypleus tridentatus juveniles and the possible strategies employed to survive on sand flats experiencing a fluctuating environment, in terms of temperature, salinity and tidal action. Activity of Tachypleus tridentatus juveniles in the field was studied on each visit between September 1997 and June 1998, during low tides when the animals were crawling at the sand surface in the intertidal zone at Shui Hau, Lantau Island, Hong Kong. The patterns of feeding trails produced by the juveniles in the field and their crawling rates were determined and compared. Juveniles began to crawl on the sand surface after the tide had receded and the substratum exposed for about 1 h. The numbers of animals observed increased and peaked after a further 1 h. The number of active individuals gradually declined as most re-burrowed when the tide returned. Juveniles generally crawled in pool areas on the sand surface. A seasonal pattern was also evident in the number of juveniles observed at the study site. Highest numbers were recorded in summer whilst only a few juveniles were seen in winter. Possible strategies adopted by the juveniles for successful survival on the sand flats are discussed. [ABSTRACT FROM AUTHOR]

Mammalian blood coagulation is based on the proteolytically induced polymerization of fibrinogens. Initially, fibrin monomers noncovalently interact with each other. The resulting homopolymers are further stabilized when the plasma transglutaminase (TGase) intermolecularly cross-links ε-(γ-glutamyl)lysine bonds. In crustaceans, hemolymph coagulation depends on the TGase-mediated cross-linking of specific plasma-clotting proteins, but without the proteolytic cascade. In horseshoe crabs, the proteolytic coagulation cascade triggered by lipopolysaccharides and b-1,3-glucans leads to the conversion of coagulogen into coagulin, resulting in noncovalent coagulin homopolymers through head-to-tail interaction. Horseshoe crab TGase, however, does not cross-link coagulins intermolecularly. Recently, we found that coagulins are cross-linked on hemocyte cell surface proteins called proxins. This indicates that a cross-linking reaction at the final stage of hemolymph coagulation is an important innate immune system of horseshoe crabs. [ABSTRACT FROM AUTHOR]

Electroretinograms were recorded from the horseshoe crab compound eye using a high-intensity light-emitting diode and a whole-eye seawater electrode. Recordings were made from both lateral eyes in natural daylight or in continuous darkness with the optic nerve intact or cut. Recordings from two eyes of the same animal in different conditions facilitated direct comparisons of the effects of diurnal lighting and circadian efferent activity on the daily patterns of sensitivity of the eye. Structural changes appear to account for about half of the total electroretinogram excursion. Circadian input begins about 45 min in advance of sunset and the nighttime sensitivity returns to the daytime values 20 min after sunrise. When the optic nerve is cut, the nighttime sensitivity shows exponential decay over the next 5 or 6 days, consistent with a light-triggered structural light adaptation process unopposed by efferent input. Our results suggest that two mechanisms mediate the increase in lateral eye sensitivity at night—physiological dark adaptation and circadian efferent input. Three mechanisms appear to be involved in mediating the decrease in lateral eye sensitivity during daylight—physiological light adaptation, a continuous structural light adaptation process, and a separate light-triggered, efferent-primed structural light adaptation process. [ABSTRACT FROM AUTHOR]

Horseshoe crab juveniles have been recorded from sand and sandy-mud nursery beaches at Pak Nai (western New Territories), San Tau and Shui Hau (Lantau Island), Hong Kong. In order to provide a better understanding of these beaches and to identify those plausible factors which have made them preferred by spawning horseshoe crabs, environmental parameters, including temperature, salinity, pH and dissolved oxygen content of the water, and particle size distribution and organic matter content of the sediments at the three sites, were determined and compared. The hydrographic, and sediment data obtained for the three study sites have revealed some common environmental features. The three nursery beaches are relatively remote, and far (in Hong Kong terms) from urbanized and densely populated areas. The beaches are generally well sheltered from strong wave action and inundated regularly by estuarine waters. Horseshoe crab adults tend to select these beaches for spawning as their protected features ensures the laid eggs are less likely to be washed out of the sand, and hatched juveniles can feed on the meiofauna and grow. Sediments of the three beaches largely comprise medium-sized sand particles and are moderately sorted, suggesting medium porosity and good water permeability. Such a sand type, with the generally high oxygen levels in incursing waters, may help create a well-oxygenated micro-environment for the normal development of horseshoe crab eggs, larvae and juveniles. Lantau Island beaches at San Tau and Shui Hau are relatively free from organic pollution, as reflected in generally high dissolved oxygen level, and low BOD and ammonia nitrogen values. Pak Nai is, however, more polluted. [ABSTRACT FROM AUTHOR]

Horseshoe crabs come ashore in attached pairs during spring high tides to mate and nest on beaches of the Atlantic and Gulf coasts. Unattached males also come ashore and crowd around the nesting pairs as satellites and engage in sperm competition with the attached male. Females with no satellites and females with large numbers of satellites nest next to one another on the same tide. When females are removed and replaced by a cement model, satellite males continue to be attracted to the same location. Models over sites where females with many satellites had nested are more attractive to males than sites from which a female with no satellites had been removed or a site where no crab had been nesting recently. A second experiment demonstrated that males are responding to chemical cues. A sponge filled with seawater taken from below a female with many satellites and placed under a model female was more attractive to males than a sponge filled with seawater. This is the first demonstration that horseshoe crabs use chemical cues, in addition to visual cues, to locate mates. [ABSTRACT FROM AUTHOR]

A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated. [ABSTRACT FROM AUTHOR]

Tachylectin-2, isolated from large granules of the hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), is a 236 amino acid protein belonging to the lectins. It binds specifically to N-acetylglucosamine and N-acetylgalactosamine and is a part of the innate immunity host defense system of the horseshoe crab. The X-ray structure of tachylectin-2 was solved at 2.0 Å resolution by the multiple isomorphous replacement method and this molecular model was employed to solve the X-ray structure of the complex with Nacetylglucosamine. Tachylectin-2 is the first protein displaying a five-bladed β-propeller structure. Five four-stranded antiparallel β-sheets of W-like topology are arranged around a central water-filled tunnel, with the water molecules arranged as a pentagonal dodecahedron. Tachylectin-2 exhibits five virtually identical binding sites, one in each β-sheet. The binding sites are located between adjacent β-sheets and are made by a large loop between the outermost strands of the β-sheets and the connecting segment from the previous β-sheet. The high number of five binding sites within the single polypeptide chain strongly suggests the recognition of carbohydrate surface structures of pathogens with a fairly high ligand density. Thus, tachylectin-2 employs strict specificity for certain N-acetyl sugars as well as the surface ligand density for self/non-self recognition. [ABSTRACT FROM AUTHOR]

When horseshoe crab embryos were treated with NaHCO at the developmental stage when the germ disc appears, multiple embryos were formed. NaHCO may effect the formation of multiple embryos by binding Ca ions of the embryo since multiple embryos were also formed by treatment with Ca free sea water. The treatment caused the blastoderm layer to tear. When the embryos were returned to normal sea water after the treatment, the blastoderm recovered. Some cell masses, probably derived from the germ disc or its prospective cells, formed during the process of the recovery. Each cell mass developed into an embryo. [ABSTRACT FROM AUTHOR]

Mitochondria from the heart of the horseshoe crab, Limulus polyphemus, contained two distinct activities of arginine kinase (AK). Approximately 50% of AK activity consisted of a fast electrophoretic form (AK) which was easily extractable from mitochondria using hypo-tonic buffers. The remaining AK activity was represented by a slow electrophoretic form (AK) which was tightly bound and could be solubilized only in the presence of non-ionic detergents. Both AK and AK appear to exist in close proximity to the mitochondrial inter-membrane space. AK was identical to cytoplasmic AK (AK) in terms of native electrophoretic mobility, subunit molecular weight, and cyanogen bromide peptide maps. In contrast, AK had a slightly higher subunit molecular weight and showed some minor, but clear-cut, differences in cyanogen bromide peptide maps from AK and AK. AK may represent a similar AK form with a hydrophobic targeting sequence for insertion into the mitochondrion and anchoring in the inner membrane. Unlike vertebrate mitochondrial creatine kinase, which binds electrostatically to the mitochondrion, a significant portion of AK in L. polyphemus mitochondria binds via hydrophobic interactions which seems to be characteristic of mitochondrial activities of phosphagen kinases in other invertebrates. [ABSTRACT FROM AUTHOR]

Tachypleus tridentatus is an endangered horseshoe crab that is found and known only in several isolated sites in southern China, and that has been suffering from the loss and fragmentation of its habitat. To evaluate its genetic diversity and population genetics, the first set of eight polymorphic microsatellite loci was developed and characterized. The number of alleles ranged from 3 to 18 per locus with the observed and expected heterozygosities ranging from 0.667 to 1.000 and from 0.543 to 0.944, respectively. These markers will be useful for the assessment of genetic variation for this species. [ABSTRACT FROM AUTHOR]

L-Gulonolactone oxidase (EC 1.1.3.8) which catalyzes oxidation of L-gulonolactone to L-ascorbic acid was detected in tissues of Limulus polyphemus. [ABSTRACT FROM AUTHOR]

The Delaware Bay contains the world's largest population of horseshoe crabs, which constitute an ecologically significant component of this estuarine ecosystem. The North Atlantic species Limulus polyphemushas an extensive geographical distribution, ranging from New Englandto the Gulf of Mexico. Recent assessments of the Delaware Bay population based on beach spawning and trawling data have suggested a considerable decrease in the number of adult animals since 1990. Considerable debate has centered on the accuracy of these estimates and their impact on marine fisheries management planning. Compounding this problem is the lack of information concerning the genetic structure of Atlantic horseshoe crab populations. This study assessed patterns of genetic variation within and between the horseshoe crab populations of Delaware Bay and Chesapeake Bay, using both Random Amplification of Polymorphic DNA (RAPD) and DNA sequence analysis of the mitochondrial cytochrome oxidase I gene (COI). We examined 41 animals from DelawareBay and 14 animals from the eastern shore of Chesapeake Bay. To provide high quality, uncontaminated genomic DNA for RAPD analysis, DNA was isolated from hemocytes by direct cardiac puncture, purified by spin column chromatography, and quantified by agarose gel electrophoresis. RAPD fingerprints revealed a relative paucity of polymorphic fragments, with generally homogeneous banding patterns both within and between populations. DNA sequence analysis of 515 bases of the 5' portion of the mitochondrial COI gene showed haplotype diversity in the Chesapeake Bay sample to be significantly higher than in the Delaware Bay sample, despite the larger size of the latter. Haplotype analysis indicates minimal contemporary gene flow between Delaware Bay and Chesapeake Bay crab populations, and further suggests that the Delaware Bay population is recovering from a recent population decline. [ABSTRACT FROM AUTHOR]

Cumulatively scored distributions of the slipper shell gastropod Crepidula sp., the sand-building polychaete worm Sabellaria vulgaris, the calcareous meandering tubed polychaete Filograna implexa, the barnacle Semibalanaus balanoides, and the lacy bryozoan Schizoporella sp. were mapped on a cm-square gridded silhouette of the prosoma, opisthosoma, and movable spines, and telson of the horseshoe crab Limulus polyphemus stranded on Reeds Beach, Cape May County, New Jersey in the Delaware Bay. Statistically tested and contoured epizoan frequencies for 81 males and 59 females revealed that slipper shells and agglutinated worm tubes are concentrated near the movable spines on the opisthosoma, but rare on recessed chilarial or opercular pleurites, the prosoma, and telson. Barnacles are disproportionately concentrated in entapophyseal pits on the opisthosoma and in the longitudinal furrows flanking the cardiac lobe of the prosoma. Bryozoans are concentrated near movable marginal spines of the opisthosoma, but also found on all areas of the prosoma, opisthosoma, and hinge area in between, as well as the telson. Different morphologic sites for their respective modal concentrations may result from competitive exclusion of bryozoansby barnacles. Genal angles have lower statistically predicted frequencies of bryozoans, barnacles, and sand worm tubes. Apertural openings of calcareous tubes are predominantly posteriorly oriented whereas the anterior margin of slipper shells are randomly oriented on the opisthosoma. Eschewing differential mortality, nonrandom distributions indicate possible: rugophilic (groove-seeking) larval settlement behavior for barnacles that preferred the recessed hinge area, longitudinal grooves, and pits; rheophilic (current-seeking) larval settlement behavior for the gastropods and tube-building polychaetes that filterfeed in the eddies generated near the movable spines of the downcurrent-sloping opisthosoma; and geotactic (gravity-influenced) larval sett [ABSTRACT FROM AUTHOR]

We studied the effects of heavy metals on the regeneration of walking legs in horseshoe crabs (Limulus polyphemus). The second walking leg was amputated in embryos (stage 20 and 21) and first instar (trilobite) larvae, and the length and morphology of the regenerated appendage was observed after molting to the second instar stage. Regeneration following continuous exposure to TBT (0.001-100 mg/L), mercury (0.001-100 mg/L), cadmium (0.01-100 mg/L), chromium (0.1-100 mg/L), lead (0.1-100 mg/L), and copper (1-100 mg/L) was measured relative to regeneration in seawater. Although regeneration was incomplete in controls, treatment with heavy metals led to smaller and/or malformed legs. The impacts of heavy metals on survival, molting, and regeneration ofhorseshoe crab larvae were ranked as follows: organotin > Hg > Cd > Cr > Zn > Pb ≧ Cu. Cu and Pb did not inhibit regeneration, even at100 mg/L. TBT, Hg, Cd, Cr, and Zn inhibited the regeneration of appendages, although first instar larvae successfully molted into second instars even after treatment. Regeneration was comparable to seawatercontrols in less than 2.5 mg/L Zn. In 5.0 and 10.0 mg/L Zn, regeneration was inhibited and the length of regenerated appendages remained shorter in all second instars. Larvae treated with 10 mg/L Zn for 1-week intervals during the molt cycle showed similar patterns of regeneration. The regeneration of claws was not all or none, and formation of the claw was proportional to the length of regenerated appendages.Limb regeneration in horseshoe crab larvae may be a useful model system for the study of pollutant impacts. [ABSTRACT FROM AUTHOR]

Lead, mercury, cadmium, chromium and manganese concentrations were measured in the eggs of horseshoe crabs (Limulus polyphemus) from 1993, 1994 and 1995; and from chelicera muscle in 1995 from Delaware Bay.Metal concentrations in the eggs represent levels derived from females. In eggs, mercury levels were below 100 ppb or were non-dectable; cadmium levels were generally low in 1993 and 1995 but were relatively higher in 1994; lead levels in eggs decreased from 558 ppb in 1993 to 87 ppm in 1995; selenium increased from 1993 to 1995; chromium decreased from 1993 to 1995; and manganese generally decreased. Leg muscles had significantly lower levels of all metals than eggs, except for mercury. [ABSTRACT FROM AUTHOR]