Limulus Polyphemus Research Articles

Cytotoxic Potential of the Novel Horseshoe Crab Peptide Polyphemusin III.

Biological activity of the new antimicrobial peptide polyphemusin III from the horseshoe crab Limulus polyphemus was examined against bacterial strains and human cancer, transformed, and normal cell cultures. Polyphemusin III has the amino acid sequence RRGCFRVCYRGFCFQRCR and is homologous to other β-hairpin peptides from the horseshoe crab. Antimicrobial activity of the peptide was evaluated and MIC (minimal inhibitory concentration) values were determined. IC50 (half-maximal inhibitory concentration) values measured toward human cells revealed that polyphemusin III showed a potent cytotoxic activity at concentrations of <10 μM. Polyphemusin III caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60, which was measured with trypan blue exclusion assay and lactate dehydrogenase-release assay. Flow cytometry experiments for annexin V-FITC/ propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antimicrobial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.

Computational biomechanical analyses demonstrate similar shell-crushing abilities in modern and ancient arthropods.

The biology of the American horseshoe crab, Limulus polyphemus, is well documented-including its dietary habits, particularly the ability to crush shell with gnathobasic walking appendages-but virtually nothing is known about the feeding biomechanics of this iconic arthropod. Limulus polyphemus is also considered the archetypal functional analogue of various extinct groups with serial gnathobasic appendages, including eurypterids, trilobites and other early arthropods, especially Sidneyia inexpectans from the mid-Cambrian (508 Myr) Burgess Shale of Canada. Exceptionally preserved specimens of S. inexpectans show evidence suggestive of durophagous (shell-crushing) tendencies-including thick gnathobasic spine cuticle and shelly gut contents-but the masticatory capabilities of this fossil species have yet to be compared with modern durophagous arthropods. Here, we use advanced computational techniques, specifically a unique application of 3D finite-element analysis (FEA), to model the feeding mechanics of L. polyphemus and S. inexpectans: the first such analyses of a modern horseshoe crab and a fossil arthropod. Results show that mechanical performance of the feeding appendages in both arthropods is remarkably similar, suggesting that S. inexpectans had similar shell-crushing capabilities to L. polyphemus This biomechanical solution to processing shelly food therefore has a history extending over 500 Myr, arising soon after the first shell-bearing animals. Arrival of durophagous predators during the early phase of animal evolution undoubtedly fuelled the Cambrian 'arms race' that involved a rapid increase in diversity, disparity and abundance of biomineralized prey species.

A review on fisheries and conservation status of Asian horseshoe crabs

Horseshoe crabs are the only extant xiphosurans and are believed to be morphologically unchanged for more than 200 million years. Of the four extant species namely, Limulus polyphemus, Tachypleus tridentatus, Tapinauchenius gigas and Carcinoscorpius rotundicauda, the latter three are found in Asian waters. Recent evidences showed that Asian horseshoe crabs are facing serious threats such as degradation of their spawning grounds and habitat, environmental pollution, overexploitation as a culinary delicacy and biomedical bleeding practices. Baseline data on the distribution and existing population of the wild horseshoe crabs remain poorly known in several Asian regions. Several studies have clearly revealed that pressure due to over-fishing of wild stock has increased tremendously in the last decade. Due to an increase in demand for Tachypleus Amebocyte Lysate (TAL) analogous to Limulus Amebocyte Lysate (LAL) in the United States, there is an urgent need to comprehensively address their fishing and conservation measures in the Asian region. This review addresses the overall studies on three species of Asian horseshoe crabs in relation to their fishing practices, local exploitation of their wild stock either for human consumption (or) by biomedical industries. The authors have structured the discussion on an international scale to address the existing problems in fishing and conservation of horseshoe crabs. Since no specific regulatory force or legislative protection act or a policy to preserve their natural stock are available to this date, this paper strongly recommends representative countries to include horseshoe crabs under their wildlife protection act to avoid further unsustainable exploitation of their wild populations.

Limulus and heart rhythm.

Great interest in the comparative physiology of hearts and their functions in Animalia has emerged with classic papers on Limulus polyphemus and mollusks. The recurrent cardiac activity-heart rate-is the most important physiological parameter and when present the kardia (Greek) is vital to the development of entire organs of the organisms in the animal kingdom. Extensive studies devoted to the regulation of cardiac rhythm in invertebrates have revealed that the basics of heart physiology are comparable to mammals. The hearts of invertebrates also beat spontaneously and are supplied with regulatory nerves: either excitatory or inhibitory or both. The distinct nerves and the source of excitation/inhibition at the level of single neurons are described for many invertebrate genera. The vertebrates and a majority of invertebrates have myogenic hearts, whereas the horseshoe crab L. polyphemus and a few other animals have a neurogenic cardiac rhythm. Nevertheless, the myogenic nature of heartbeat is precursor, because the contraction of native and stem-cell-derived cardiomyocytes does occur in the absence of any neural elements. Even in L. polyphemus, the heart rhythm is myogenic at embryonic stages.

Illustrations for Health Assessment Techniques of the Atlantic Horseshoe Crab, Limulus polyphemus

Horseshoe crab blood cells detect extremely small amountsof bacteria allowing them to be used in medicine to test thesterilization of medical devices, vaccines, and implants. This is aworldwide standard test called Limulus Amebocyte Lysate. TheNational Aquarium in Baltimore, MD, has developed guidelinesfor health examinations that are aimed to improve healthmaintenance and conservation strategies for horseshoe crabs.Illustrations were created to accompany this specic literatureto ensure the overall eectiveness of the material. A total of 28color illustrations were created to help educate veterinariansand veterinarian technicians, aquarists, and biologists.

Biological activity of LALF32-51–E7, a vaccine candidate for the treatment of anogenital lesions associated to HPV16

Cervical cancer is the second leading cause of cancer death in women. It is well established that human papillomavirus (HPV) is the cause of virtually 100% of these cancers, 70% of which are attributable to infections with HPV types 16 or 18. We developed a fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein (LALF32-51) linked to the HPV16 E7 antigen (LALF32-51–E7) that is obtained from Escherichia coli. The development of new treatments as mentioned requires a robust and reproducible procedure for batch release. We used the combination of three batches of LALF32-51-E7 with Al(OH)3 in an experimental design to develop a biological activity test for batch release of this candidate, which uses prophylactic immunization in the TC-1 tumor mouse model. Two injections, spaced for 7 days with LALF31-52-E7+Al(OH)3 are sufficient for inducing a potent anti-tumor response in the TC-1 tumor model. LALF31-52-E7 vaccine candidates in combination with Al(OH)3, exhibited in C57BL/6 mice over 86% protection against tumor challenge and high titers of specific antibodies against LALF31-52-E7. The results obtained across three batches of LALF32-51–E7+Al(OH)3 with this simple and uncomplicated assay based on prophylactic immunization in the TC-1 mouse model suggest that it would be feasible to implement it as a biological activity assay for the release of production batches of LALF32-51–E7.

Assessing the movements of American horseshoe crabs (Limulus polyphemus) around a marine protected area in Cape Cod, MA, USA

Marine protected areas (MPAs) can be useful for conservation efforts, yet the effectiveness of MPAs relies on their size and location, as well as the spatial ecology of the focal species. We investigated the effectiveness of a small MPA adjacent to Cape Cod, MA (USA) for protecting American horseshoe crabs (Limulus polyphemus). We used a fixed acoustic telemetry array to study the spatial ecology of 24 adult horseshoe crabs off the coast of Chatham, Massachusetts, from June 2010 to November 2011. Two groups of horseshoe crabs were tagged in spawning habitats (separated by ∼2.0 km) with differing commercial harvesting pressure: one group inside Stage Harbor where harvesting is permitted, and the other within Monomoy National Wildlife Refuge and the Cape Cod National Seashore (the MPA) where harvesting is prohibited. In the first year of study, crabs tagged in the MPA did not go to Stage Harbor, but instead remained in the MPA and adjacent high-energy beach, whereas crabs tagged in Stage Harbor moved throughout the entire study area. In the second year of study, crabs from both tagging locations used all areas. All crabs generally moved greater distances in the MPA than in other areas, and the majority of movement we recorded was in late spring and summer. All crabs that we tagged used similar areas around Cape Cod. The MPA only provides protection when crabs are in that area, which may only be for a few months of the year. Given that all crabs used Stage Harbor, they were all exposed to potential harvest, but the MPA did provide some protection for all crabs.

The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sustainability

Every year the Atlantic horseshoe crab (Limulus polyphemus) arrives on shore to spawn, a sight once taken for granted. However, in addition to the gradual climate changes impacting all ecosystems, commercial demand from the widespread application of Atlantic horseshoe crab blood in industrial endotoxin testing and steady use as eel and whelk bait has brought the future of this enduring species into question. In response, regulations have been adopted to enhance the traceability and record keeping of horseshoe crab harvest, which has historically been difficult to track. However, these regulations do not restrict or limit LAL harvest in any significant manner. Still, sometimes-lethal biomedical bleeding process and associated behavioral changes pose a risk to horseshoe crab viability after bleeding and once returned to the waters. As a result, regulators and environmentalists are concerned that current trends and overfishing of this marine arthropod will significantly impact the surrounding ecosystem. This review examines their role and recent trends in the biomedical industry that are impacting these ancient creatures and the derivative effect on shorebirds, while considering emerging alternatives where feasible, as well as ways to ensure sustainable and pragmatic harvesting strategies. Ultimately, healthy populations of horseshoe crabs are vital to restoring and maintaining ecosystems while balancing the need for medical and research applications entirely dependent on these unique creatures.

The mitochondrial genome of the oribatid mite Paraleius leontonychus: new insights into tRNA evolution and phylogenetic relationships in acariform mites

Bilaterian mitochondrial (mt) genomes are circular molecules that typically contain 37 genes. To date, only a single complete mitogenome sequence is available for the species-rich sarcoptiform mite order Oribatida. We sequenced the mitogenome of Paraleius leontonychus, another species of this suborder. It is 14,186 bp long and contains 35 genes, including only 20 tRNAs, lacking tRNAGly and tRNATyr. Re-annotation of the mitogenome of Steganacarus magnus increased the number of mt tRNAs for this species to 12. As typical for acariform mites, many tRNAs are highly truncated in both oribatid species. The total number of tRNAs and the number of tRNAs with a complete cloverleaf-like structure in P. leontonychus, however, clearly exceeds the numbers previously reported for Sarcoptiformes. This indicates, contrary to what has been previously assumed, that reduction of tRNAs is not a general characteristic for sarcoptiform mites. Compared to other Sarcoptiformes, the two oribatid species have the least rearranged mt genome with respect to the pattern observed in Limulus polyphemus, a basal arachnid species. Phylogenetic analysis of the newly sequenced mt genome and previously published data on other acariform mites confirms paraphyly of the Oribatida and an origin of the Astigmata within the Oribatida.

Habitat risk: Use of intertidal flats by foraging red knots (Calidris canutus rufa), ruddy turnstones, (Arenaria interpres), semipalmated sandpipers (Calidris pusilla), and sanderling (Calidris alba) on Delaware Bay beaches

Shorebirds usually forage on intertidal flats that are exposed during low tide, and roost on higher areas when the tidal flats are covered with water. During spring migration on Delaware Bay (New Jersey) shorebirds mainly forage on horseshoe crab (Limulus polyphemus) eggs that are concentrated at the high tide line. However, they also use other habitats for foraging. We examined habitat use of 4 species of shorebirds (with declining populations) at five Delaware Bay beaches to determine their use of the intertidal habitat (2015, 2016). We observed birds in three sections at different distances from the mean high tideline (< 100 m, 101–200 m, and 201–300 m)ne. We examined the presence of red knots (Calidris canutus rufa), ruddy turnstones (Arenaria interpres), semipalmated sandpipers (Calidris pusilla), and sanderling (Calidris alba) as a function of date, tide cycle, section shorebirds foraged from the mean high tide line, and presence of other shorebird species. Understanding how these species use the intertidal flats is important because these habitats are at risk from coastal development, sea level rise, and decreases in intertidal space, including the possible expansion of intertidal oyster culture. Overall, knots were present in the intertidal on 67% of the surveys, turnstones were present on 86% of the surveys, semipalmated sandpipers were present on 77% of the surveys, and sanderling were present on 86% of the surveys. Use of the intertidal flats varied among beaches. Peak and mean numbers of shorebirds/ decreased in each census section, as distance to mean high tideline increased. In general, shorebirds foraged at the waters’ edge during high tide, and then moved out onto the intertidal flats. The strongest interspecific associations were between red knots and ruddy turnstones, and the lowest associations were between sanderling and semipalmated sandpipers. Variations in numbers of each species in 2016 were mainly explained by the number of other species, section (distance from the mean high tide line), location (one of 5 beaches), and date for all species (and minutes to low tide for sanderling). These data indicate that these 4 species use intertidal flats as they become available, and that the mean number in each newly exposed census section of the flats may be lower than in the previous one, partly as a result of some birds remaining in each previously-exposed section. We discuss the management and regulatory implications of shorebird use of the intertidal flats, which include protection of high quality intertidal for foraging by shorebirds.

Hemocyanin-derived phenoloxidase reaction products display anti-infective properties

Hemocyanin is a multi-functional protein located in the hemolymph (blood) of certain arthropods and molluscs. In addition to its well-defined role in oxygen transport, hemocyanin can be converted into a phenoloxidase-like enzyme. Herein, we tested the antimicrobial properties of horseshoe crab (Limulus polyphemus) hemocyanin-derived phenoloxidase reaction products using broad ranges of phenolic substrates (e.g. l-DOPA) and microbial targets (Gram-positive/negative bacteria, yeast). The enzyme-catalysed turnover of several substrates generated (by)products that reduced significantly the number of colony forming units. Microbicidal effects of hemocyanin-derived phenoloxidase were thwarted by the inhibitor phenylthiourea. Data presented here further support a role for hemocyanin in invertebrate innate immunity.

Horseshoe Crab (Limulus polyphemus) Movements Following Tagging in the Delaware Inland Bays, USA

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.

A new lipid carrier protein in the cattle tick Rhipicephalus microplus

Tick infestation in cattle reflects the main cause of economic loss to cattle producers. This is due to several reasons but mainly to their ability to feed on blood and generate a huge amount of eggs. Lipid transport in arthropods is achieved by highly specialized hemolymphatic lipoproteins, which resemble those described in vertebrate blood. Such lipoproteins continuously deliver lipids through the blood to growing eggs. The injection of radioactive [3H] palmitic acid into tick hemocoel showed that the gut, ovary, fat body and Gene’s organ were the main organs of incorporation of this labeled fatty acid. The rate of [3H] palmitic acid incorporation by the organs was high up to 30 min after injection. The [3H] palmitic acid incorporated by these organs was later found in phospholipids and neutral lipids. Here, we describe the purification and characterization of a key player of lipid dynamics in tick hemolymph. The Rhipicephalus microplus lipid-apolipoprotein complex (RmLCP) is a new high-density lipoprotein (1.18 g/mL), which accounts for over 90% of [3H] palmitic acid present in the hemolymph. It has a native molecular weight of 420 kDa and is composed of one subunit of 122 kDa. Protein identification analysis of RmLPC subunit showed two better hits: vitellogenin 2 (23% protein coverage) and vitellogenin 5 (29% protein coverage), respectively and similarities with hemolymphatic apolipoproteins of arachnids such as the tick Ixodes scapularis (80%), the mite Galendromus occidentalis (44%) and the spider Parasteatoda tepidariorum (43%) and also for the insects Locusta migratoria (45%), Drosophila melanogaster (42%) and Manduca sexta (47%) to vitellogenin 2 and tick Ixodes scapularis (83%), the crab Limulus polyphemus (55%) and the oyster Crassostrea gigas (55%) to vitellogenin 5. Furthermore, it shows a distinct lipid composition from most arthropod lipoproteins, being composed of 40% free cholesterol, 27% phospholipids, 20% triacylglycerol and 15% hydrocarbons. In addition to binding most hemolymphatic fatty acids, this lipoprotein also binds and transports free cholesterol. In conclusion, the present study provides insight into the macromolecules involved in arachnid metabolism, which have significant potential for future use for the biological control of ticks.

A 3D anatomical atlas of appendage musculature in the chelicerate arthropod Limulus polyphemus.

Limulus polyphemus, an archetypal chelicerate taxon, has interested both biological and paleontological researchers due to its unique suite of anatomical features and as a useful modern analogue for fossil arthropod groups. To assist the study and documentation of this iconic taxon, we present a 3D atlas on the appendage musculature, with specific focus on the muscles of the cephalothoracic appendages. As L. polyphemus appendage musculature has been the focus of extensive study, depicting the muscles in 3D will facilitate a more complete understanding thereof for future researchers. A large museum specimen was CT scanned to illustrate the major exoskeletal features of L. polyphemus. Micro-CT scans of iodine-stained appendages from fresh, non-museum specimens were digitally dissected to interactively depict appendage sections and muscles. This study has revealed the presence of two new muscles: one within the pushing leg, located dorsally relative to all other patella muscles, and the other within the male pedipalp, located in the modified tibiotarsus. This atlas increases accessibility to important internal and external morphological features of L. polyphemus and reduces the need for destructive fresh tissue dissection of specimens. Scanning, digitally dissecting, and documenting taxa in 3D is a pivotal step towards creating permanent digital records of life on Earth.

Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus.

Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.

The ontogeny of Limulus polyphemus (Xiphosura s. str., Euchelicerata) revised: looking "under the skin".

In recent years, methods for investigating the exo-morphology of zoological specimens have seen large improvements. Among new approaches, auto-fluorescence imaging offers possibilities to document specimens under high resolution without introducing additional artifacts as, for example, seen in scanning electron microscopy (SEM) imaging. Additionally, while SEM imaging is restricted to the outer morphology of the current instar, auto-fluorescence imaging can be used to document changes of the outer morphology of the next instar underneath the cuticle of the current instar. Thus, reinvestigating seemingly well known species with these methods may lead to interesting new insights. Here we reinvestigate the late embryonic development of the xiphosuran ("sword tail") Limulus polyphemus, which is often treated as a proxy for early eucheliceratan evolution. In addition to entire specimens, the appendages of the embryos were dissected off and documented separately with composite-autofluorescence microscopy. Based on these data, we can distinguish six developmental stages. These stages do not match exactly the formerly described stages, as these were largely based on SEM investigation. Our stages appear to represent earlier or later phases within what has in other studies been identified as one stage. This finer subdivision is visible as we can see the developing cuticle under the outer cuticle. In comparison to data from fossil xiphosurans, our results and those of other studies on the ontogeny of L. polyphemus point to a derived mode of development in this species, which argues against the idea of L. polyphemus as a "living fossil."

Use of intertidal habitat by four species of shorebirds in an experimental array of oyster racks, reefs and controls on Delaware Bay, New Jersey: Avoidance of oyster racks

Many shorebirds breed in Arctic habitats, and migrate south to wintering quarters in the Southern Hemisphere. Shorebirds mainly forage on intertidal mudflats at low tide. A key spring stopover for shorebirds in North America is Delaware Bay, New Jersey, where shorebirds feed on horseshoe crab (Limulus polyphemus) eggs at high tide. The importance of intertidal to migrant shorebirds has been overlooked. This paper examines foraging of 4 shorebird species at Reeds Beach, Delaware Bay. The intertidal zone was divided into an experimental array with oyster racks, artificial reefs, and controls to quantify the number of birds present in each section. The best models for all species (except sanderling, Calidris alba) explained over 60% of variation in number present as a function of other shorebirds, date, and treatment. Time of day was the only factor affecting the number of sanderlings. Date is a contributing factor because numbers are low in early May, peak by May 20–25th, and decrease in late May as shorebirds leave for the Arctic. Red knots (Calidris canutus rufa), turnstones (Arenaria interpres), and semipalmated sandpipers (Calidris pusilla) were present less often, and in smaller numbers in the oyster rack treatment (compared to the other treatments). There was a high association between foraging species. The data clearly show avoidance of the oyster racks by knots, turnstones and semipalmated sandpipers, but they did not avoid those with reefs or the control. These results are important because of the desire to expand aquaculture along the Delaware Bay into prime shorebird foraging areas. The Precautionary Principle dictates that the expansion of intertidal oyster culture be halted in areas of high foraging value for red knots (threatened in the U.S.) and other shorebirds until any effects on fitness are determined.

The gnathobasic spine microstructure of recent and Silurian chelicerates and the Cambrian artiopodan Sidneyia: Functional and evolutionary implications

Gnathobasic spines are located on the protopodal segments of the appendages of various euarthropod taxa, notably chelicerates. Although they are used to crush shells and masticate soft food items, the microstructure of these spines are relatively poorly known in both extant and extinct forms. Here we compare the gnathobasic spine microstructures of the Silurian eurypterid Eurypterus tetragonophthalmus from Estonia and the Cambrian artiopodan Sidneyiainexpectans from Canada with those of the Recent xiphosuran chelicerate Limulus polyphemus to infer potential variations in functional morphology through time. The thickened fibrous exocuticle in L. polyphemus spine tips enables effective prey mastication and shell crushing, while also reducing pressure on nerve endings that fill the spine cavities. The spine cuticle of E. tetragonophthalmus has a laminate structure and lacks the fibrous layers seen in L. polyphemus spines, suggesting that E. tetragonophthalmus may not have been capable of crushing thick shells, but a durophagous habit cannot be precluded. Conversely, the cuticle of S. inexpectans spines has a similar fibrous microstructure to L. polyphemus, suggesting that S. inexpectans was a competent shell crusher. This conclusion is consistent with specimens showing preserved gut contents containing various shelly fragments. The shape and arrangement of the gnathobasic spines is similar for both L. polyphemus and S. inexpectans, with stouter spines in the posterior cephalothoracic or trunk appendages, respectively. This differentiation indicates that crushing occurs posteriorly, while the gnathobases on anterior appendages continue mastication and push food towards and into the mouth. The results of recent phylogenetic analyses that considered both modern and fossil euarthropod clades show that xiphosurans and eurypterids are united as crown-group euchelicerates, with S. inexpectans placed within more basal artiopodan clades. These relationships suggest that gnathobases with thickened fibrous exocuticle, if not homoplasious, may be plesiomorphic for chelicerates and deeper relatives within Arachnomorpha. This study shows that the gnathobasic spine microstructure best adapted for durophagy has remained remarkably constant since the Cambrian.

Effects of a Beach Nourishment Project in Jamaica Bay, New York, on Horseshoe Crab (Limulus polyphemus) Spawning Activity and Egg Deposition

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.

Melanization in response to wounding is ancestral in arthropods and conserved in albino cave species

Many species adapted to aphotic subterranean habitats have lost all body pigmentation. Yet, melanization is an important component of wound healing in arthropods. We amputated appendages in a variety of cave-adapted and surface-dwelling arthropods. A dark clot formed at the site of injury in most species tested, including even albino cave-adapted species. The dark coloration of the clots was due to melanin deposition. The speed of wound melanization was uncorrelated with a difference in metabolic rate between surface and cave populations of an amphipod. The chelicerate Limulus polyphemus, all isopod crustaceans tested, and the cave shrimp Troglocaris anophthalmus did not melanize wounds. The loss of wound melanization in T. anophthalmus was an apomorphy associated with adaptation to subterranean habitats, but in isopods it appeared to be a symplesiomorphy unrelated to colonization of subterranean habitats. We conclude that wound melanization i) is an important part of innate immunity because it was present in all major arthropod lineages, ii) is retained in most albino cave species, and iii) has been lost several times during arthropod evolution, indicating melanization is not an indispensable component of wound healing in arthropods.