Search Results (31)

Accurate detection of microbial contamination is essential in the assessment of toxicological and immunological responses to various materials, as low-level contaminants can lead to confounding results. Traditional endotoxin testing relies on the Limulus Amebocyte Lysate (LAL) assay, which depends on horseshoe crab blood and raises both ecological and ethical concerns. Sustainable alternatives such as recombinant Factor C (rFC) provide a promising solution, yet validation for the detection of endotoxin in nanomaterials remains incomplete. In this study, we have used rFC alongside Toll-like receptor (TLR) reporter assays to detect both endotoxin and broader microbial contaminants in 31 nanomaterials from diverse classes. Special attention was given to assay interference by nanomaterials to ensure reliable detection. The rFC assay demonstrated a sensitive detection limit of 0.005 EU/mL, equivalent to the LAL assay, and showed that more than 50% of tested nanomaterials contained low-level endotoxin contamination. Additionally, several nanomaterials activated the TLR2 reporter, indicative of microbial contaminants beyond endotoxin. These results suggest that rFC can serve as a sustainable and reliable replacement for LAL in nanomaterial endotoxin testing but also emphasize the limitations of relying solely on endotoxin-specific assays. We recommend that future nanotoxicological evaluations integrate rFC with complementary methods, such as TLR-based approaches, and include thorough interference controls to ensure robust and comprehensive microbial contamination assessment. Full article

The mortality rate of first- to second-instar horseshoe crabs during molting is extremely high under culture conditions (pH of 7.6 ± 0.1, salinity of 27 ± 2, temperature of 26–32 °C), and we preliminarily speculate that it is related to disease. Our team found that Ectoplana limuli was attached to the ventral limbs of adult horseshoe crabs during culture. Parasite samples were collected from the external appendages and mouthparts of adult Tachypleus tridentatus for classification and identification. The primary objective of this experiment was to identify the species of this parasite and determine its taxonomic status. To this end, the experiment employed a combination of morphological methods and 18S rDNA gene molecular markers. The obtained sequences showed over 99% homology with Ectoplana limuli. Sequence alignment and phylogenetic tree results indicated that Ectoplana limuli showed a closer genetic relationship with Nerpa fistulata, but more distant relationships with Paucumara and Baikalobia. This is the first time that this parasite has been found in China, providing additional information for the study of horseshoe crab diseases. Full article

Selenium (Se) is an important trace element in our body; however, food composition data remain limited due to analytical challenges and interferences. Seafood, abundant in Thailand, is recognized as a rich source of Se. This study aimed to expand knowledge on Se content in seafood prepared using traditional Thai cooking methods. Twenty seafood species were selected and prepared by boiling, frying, and grilling. Inductively Coupled Plasma–Triple Quadrupole–Mass Spectrometry (ICP-MS/MS) was used to analyze total Se contents in selected seafood species. Results revealed significant variation in Se content across species and cooking methods. The Indo-Pacific horseshoe crab showed the highest Se concentration, with fried samples reaching 193.9 μg/100 g. Se concentrations were in the range of 8.6–155.5 μg/100 g (fresh), 14.3–106.6 μg/100 g (boiled), 17.3–193.9 μg/100 g (fried), and 7.3–160.1 μg/100 g (grilled). Results found significant effects of species and cooking method on Se content (p < 0.05). Fried seafood exhibited the highest estimated marginal mean Se concentration (a 78.8 μg/100 g edible portion), significantly higher than other methods. True retention (%TR) of Se ranged from 40.4% to 100%, depending on species and method. Bigfin reef squid, wedge shell, and silver pomfret showed the highest %TR (100%), while splendid squid exhibited the lowest (52.5%). Significant interaction effects on %TR were also observed (p < 0.05). Fried seafood had the highest mean %TR (88.8%), followed by grilled (82.1%) and boiled (79.7%). These findings highlight the effects of both species and cooking method on Se retention, emphasizing the nutritional value of selected seafood in preserving bioavailable Se after cooking. Full article

Limulus amoebocyte lysate (LAL) assays have emerged as among the most effective approaches for detecting endotoxins and fungi in vitro since they were first tested 50 years ago. Although detailed protocols are publicly available, conventional LAL collection methods (3% sodium chloride) waste as much as 80% of the total LAL during blood accumulation, confirming the incompatibility of these methods with the lasting survival of the American horseshoe crab. For this reason, new implementations of blood collection–suspension buffer combinations are critical. Here, we evaluated the ability of different blood collection solutions to inhibit exocytosis and subsequently treated the cells with CaCl₂ to stimulate exocytosis and improve the yield of LAL. Two test methods, chromogenic and turbidimetric tests for LAL activity, were evaluated. Crabs were bled during the bleeding season. The crab blood samples were collected with the following blood collection solutions: citric acid buffer, malic acid buffer, PBS buffer, and PBS–caffeine buffer. The cell pellets were washed with 3% NaCl and subsequently resuspended in LRW or CaCl₂ to facilitate degranulation. Both the chromogenic test and the turbidimetric assay were used to evaluate the LAL enzyme activity. Citric acid buffer, malic acid buffer, PBS buffer, and PBS–caffeine buffer blocked exocytosis, resulting in the high yields of LAL. There was no observable effect on the activity output of crab size via a chromogenic test with PBS–caffeine buffer during the bleeding season. This protocol substantially benefited prior processes, as the PBS–caffeine collection mixture decreased amoebocyte aggregation/clot formation during processing. Furthermore, we evaluated the specific biochemical parameters of PBS–caffeine-derived LAL. We developed an accessible, promising phosphate–caffeine-based blood collection buffer that prevents amoebocyte degranulation during blood collection, maximizing the LAL yield. Moreover, our analysis revealed that phosphate–caffeine-derived LAL is uniquely adaptable to compatibility with chromogenic and turbidimetric assay techniques. By employing this method for LAL blood extraction, our same-cost approach fostered significantly greater LAL yields, simultaneously ensuring a healthy limulus polyphemus population. Full article

The Cambrian–Ordovician Plankton Revolution played a crucial role in the Great Ordovician Biodiversification Event (GOBE) or Ordovician Radiation, as a driver of diversification. The emergence of new planktonic species enhanced primary productivity and improved nutrient cycling, fueling diversification across trophic levels. In this context, established plankton consumers like sponges and cnidarians thrived, and animals like euarthropods also radiated in response to these environmental conditions. Here, we hypothesize that Offacolidae, a small group of early chelicerates (the group including sea spiders, spiders, mites, and horseshoe crabs) known from the early Ordovician to the end of the Silurian, were suspension feeders that diversified within this changing ecosystem. Extant chelicerates are primarily predators or parasites, with no known cases of suspension feeding, which is also the case in extinct members. However, anatomical and environmental evidence suggest that Offacolidae may have adopted this feeding strategy. We examine the environmental conditions in which Offacolidae fossils were found, considering both biotic and abiotic factors such as paleolatitude, bathymetry, and the associated plankton community. We also assess the possible biomechanics of their unique appendages to determine their suitability for suspension feeding. Finally, comparisons with extant arthropods, such as the suspension-feeding crustaceans Limnomysis benedeni, Atya gabonensis, Subeucalanus pileatus, and the genus Emerita, provide insights into possible evolutionary analogies in their morphology, which may have served the same function. If confirmed, this hypothesis would make Offacolidae a unique case within chelicerates, illustrating how exceptional early Ordovician conditions fostered novel ecological adaptations and highlighting an interesting case of analogy between different lineages of euarthropods. Full article

Xiphosurids (Merostomata, Xiphosura) are a group of chelicerates with a rich and complex evolutionary history that is constantly being updated through new discoveries. In this study, we re-estimated the divergence time of the extant horseshoe crab species with new fossil calibration points and addressed the inter- and intraspecific relationships of the American horseshoe crab through a phylogeographic perspective. In order to achieve our objectives, three datasets were compiled from fragments of different lengths of the COI gene that include sequences from 154 individuals, representing the Mexican populations. In addition to these, the datasets also included previously published sequences corresponding to individuals from different US populations and Asian horseshoe crab species. Firstly, we estimated the divergence times of extant horseshoe crab species by Bayesian methods using multiple fossil calibration points. Subsequently, we investigated the phylogeographic relationships and demographic history of Limulus polyphemus in the Americas utilizing various datasets. The time of divergence of the two Asian species clades was estimated to be approximately 127 million years ago (Ma). Phylogeographic relationships between the Asian and American species are linked through a minimum of 86 mutational steps. In America, phylogeographic relationships reflect differentiation between US and Mexican populations of L. polyphemus. We detect signs of demographic expansion for the Mexican population during the last 75,000 years, as well as an absence of phylogeographic structuring. The evolutionary history of horseshoe crabs is older than previously believed; however, the current distribution and demographic changes have probably been influenced by environmental events of the recent past, such as the glacial–interglacial periods that occurred during the Pleistocene. Full article

This paper proposes an underwater image enhancement technology based on an artificial lateral inhibition network (ALIN) generated in the compound eye of a bionic horseshoe crab robot (BHCR). The concept of a horizontal suppression network is applied to underwater image processing with the aim of achieving low energy consumption, high efficiency processing, and adaptability to limited computing resources. The lateral inhibition network has the effect of “enhancing the center and suppressing the surroundings”. In this paper, a pattern recognition algorithm is used to compare and analyze the images obtained by an artificial lateral inhibition network and eight main underwater enhancement algorithms (white balance, histogram equalization, multi-scale Retinex, and dark channel). Therefore, we can evaluate the application of the artificial lateral inhibition network in underwater image enhancement and the deficiency of the algorithm. The experimental results show that the ALIN plays an obvious role in enhancing the important information in underwater image processing technology. Compared with other algorithms, this algorithm can effectively improve the contrast between the highlight area and the shadow area in underwater image processing, solve the problem that the information of the characteristic points of the collected image is not prominent, and achieve the unique effect of suppressing the intensity of other pixel points without information. Finally, we conduct target recognition verification experiments to assess the ALIN’s performance in identifying targets underwater with the BHCR in static water environments. The experiments confirm that the BHCR can maneuver underwater using multiple degrees of freedom (MDOF) and successfully acquire underwater targets. Full article

Trilobites, as typical euarthropods, possess compound eyes. In 1901, Lindström was the first to describe them in detail; on the one hand, we reconsider his descriptions of the different modes of trilobite eyes; on the other hand, we expand this by compiling the observations that have been possible in recent years. There are two, perhaps three kinds of trilobite compound eyes. The first are the primordial holochroal eyes, which are actually apposition compound eyes, similar to those of many modern diurnal crustaceans and insects. The abathochroal eyes, often referred to as the second form, are probably a subtype of the holochroal eyes. Consequently, the second is the schizochroal eye of phacopid trilobites, which are hyper-compound eyes composed of numerous small compound eyes below each of the big lenses, which appear from outside as one big lateral eye each. Thirdly, one may call the maculae light-sensitive organs, but this is still uncertain. Comparing what are probably the oldest trilobite eyes described so far with other forms, it is possible to conclude that the sensory apparatus is much older than the fossil record of trilobite eyes and probably developed in Precambrian times. The refractive apparatus, however, was developed later and separately within the systematic groups. This explains why, for example, the mandibulates have a lens and a crystalline cone. Still, the chelicerate xiphosurans, such as horseshoe crabs or eurypterids, possess a lens cylinder with an index gradient but no crystalline cone. Furthermore, this can explain why the calcite character of trilobites is unique in the arthropod kingdom. An important discovery is the probably epidermal, lens-building cells encompassing a prospective lens of Schmidtiellus reetae Bergström 1973 from the early Lower Cambrian of Estonia. We reconsider the morphology of hypostome maculae and interpret them as a potential phylogenetic relict and a potential predecessor of all arthropod ommatidial compound eyes. It will be of great relevance for future research to understand the evolution of compound eyes and vision because we witness the emergence of the first lenses in the trilobite, if not the arthropod kingdom. Full article

The venoms of Theraphosidae spiders have evolved into diverse natural pharmacopeias through selective pressures. Cryptococcus neoformans is a global health threat that frequently causes life-threatening meningitis and fungemia, particularly in immunocompromised patients. In this study, we identify a novel anti-C. neoformans peptide, QS18 (QCFKVCFRKRCFTKCSRS), from the venom gland of China’s native spider species Chilobrachys liboensis by utilizing bioinformatic tools. QS18 shares over 50% sequence similarity with tachyplesin peptides, previously identified only in horseshoe crab hemocytes, expanding the known repertoire of the tachyplesin family to terrestrial arachnids. The oxidative folding of QS18 notably enhances its antifungal activity and stability, resulting in a minimum inhibitory concentration of 1.4 µM. The antimicrobial mechanism of QS18 involves cell membrane disruption. QS18 exhibits less than 5% hemolysis in human erythrocytes, indicating microbial selectivity and a favorable safety profile for therapeutic use. Furthermore, mouse model studies highlight QS18’s ability as an antifungal agent with notable anti-inflammatory activity. Our study demonstrates QS18 as both a promising template for spider venom peptide research and a novel candidate for the development of peptide antifungals. Full article

Horseshoe crabs represent the most ancestral chelicerate lineage characterized by marine ecology and the possession of lateral compound eyes. While considered living fossils, recent studies reported an unusual number of Pax6 genes in the Atlantic horseshoe crab Limulus polyphemus. Pax genes encode ancient metazoan transcription factors, which comprise seven subfamilies. Among these, the members of the Pax6 subfamily confer critical functions in the development of the head, the visual system, and further body plan components. Arthropods are generally characterized by two Pax6 subfamily homologs that were discovered in Drosophila and named eyeless (ey) and twin of eyeless (toy). However, whole genome sequence searches uncovered three homologs of ey and two homologs of toy in L. polyphemus. These numbers are explained by the occurrence of likely three whole genome duplications in the lineage to the last common ancestor of L. polyphemus and the three additional members of the extant horseshoe crab species cluster. Here, we report that all five L. polyphemus Pax6 paralogs are conserved in the approximately 135-million-year-old horseshoe crab species cluster and that they evolve under strong purifying selection. Largely homogenous protein sequence diversification rates of ey and toy paralogs suggest subfunctionalization as the likeliest preservation trajectory. However, our studies further revealed evidence that the horseshoe crab ey1 and ey2 paralogs share a derived splice isoform that encodes a unique five amino acid-long insertion in helix 3 of the homeodomain. This suggests that the exceptional expansion of the horseshoe crab Pax6 gene family repertoire was also associated with regulatory diversification and possibly innovation. Full article

The elements in blood normally reflect the levels in prey, indicating a recent exposure. Laughing gulls (Leucophaes atricilla) eat mainly horseshoe crab eggs (Limulus polyphemus) in the spring in Delaware Bay, New Jersey. The levels of arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), and selenium (Se) in the blood of laughing gulls foraging on crab eggs were examined in Delaware Bay to provide information on a species that is normally a generalist, and to determine if the levels of these elements were similar in 2019 and 2022/2023, were intercorrelated, and were related to those in crab eggs. Hg increased from 2019 (136 ± 31 ng/g) to 2022/2023 (473 ± 75 ng/g), while Cd and Se decreased. There were some significant correlations among elements and a close relationship between the element levels in blood and those in crab eggs collected in the same month (except for As). The levels differed between laughing gulls and three species of shorebirds for As and Cd. The elements in the blood of gulls and shorebirds should be similar because they eat mainly the same eggs in the same places. A significant proportion of laughing gull blood samples had levels of Hg and Se that were above the levels associated with adverse effects, which requires further examination. Full article

Pain in response to tissue damage functions to change behaviour so that further damage is minimised whereas healing and survival are promoted. This paper focuses on the behavioural criteria that match the function to ask if pain is likely in the main taxa of arthropods. There is evidence consistent with the idea of pain in crustaceans, insects and, to a lesser extent, spiders. There is little evidence of pain in millipedes, centipedes, scorpions, and horseshoe crabs but there have been few investigations of these groups. Alternative approaches in the study of pain are explored and it is suggested that studies on traumatic mating, agonistic interactions, and defensive venoms might provide clues about pain. The evolution of high cognitive ability, sensory systems, and flexible decision-making is discussed as well as how these might influence the evolution of pain-like states. Full article

Understanding variations in metal levels in biota geographically and under different environmental conditions is essential to determining risk to organisms themselves and to their predators. It is often difficult to determine food chain relationships because predators may eat several different prey types. Horseshoe crab (Limulus polyphemus) eggs form the basis for a complex food web in Delaware Bay, New Jersey, USA. Female horseshoe crabs lay thumb-sized clutches of eggs, several cm below the surface, and often dislodge previously laid eggs that are brought to the surface by wave action, where they are accessible and critical food for migrant shorebirds. This paper compares metal and metalloid (chromium [Cr], cadmium [Cd], lead [Pb], mercury [Hg], arsenic [As] and selenium [Se]) concentrations in horseshoe crab eggs collected on the surface with concentrations in eggs from clutches excavated from below the sand surface, as well as examining metals in eggs from different parts of the Bay. The eggs were all collected in May 2019, corresponding to the presence of the four main species of shorebirds migrating through Delaware Bay. These migrating birds eat almost entirely horseshoe crab eggs during their stopover in Delaware Bay, and there are differences in the levels of metals in blood of different shorebirds. These differences could be due to whether they have access to egg clutches below sand (ruddy turnstones, Arenaria interpres) or only to eggs on the surface (the threatened red knot [Calidris canutus rufa] and other species of shorebirds). Correlations between metals in clutches were also examined. Except for As and Cd, there were no significant differences between the metals in crab egg clutches and eggs on the surface that shorebirds, gulls, and other predators eat. There were significant locational differences in metal levels in horseshoe crab eggs (except for Pb), with most metals being highest in the sites on the lower portion of Delaware Bay. Most metals in crab eggs have declined since studies were conducted in the mid-1990s but were similar to levels in horseshoe crab eggs in 2012. The data continue to provide important monitoring and assessment information for a keystone species in an ecosystem that supports many species, including threatened and declining shorebird species during spring migration. Full article

Carcinoscorpius rotundicauda (C. rotundicauda) is one of the four species of horseshoe crabs (HSCs). The HSC hemocytes store defense molecules that are released upon encountering invading pathogens. The HSCs rely on this innate immunity to continue its existence as a living fossil for more than 480 million years. To gain insight into the innate mechanisms involved, transcriptomic analysis was performed on isolated C. rotundicauda hemocytes challenged with lipopolysaccharides (LPS), the main components of the outer cell membrane of gram-negative bacteria. RNA-sequencing with Illumina HiSeq platform resulted in 232,628,086 and 245,448,176 raw reads corresponding to 190,326,253 and 201,180,020 high-quality mappable reads from control and LPS-stimulated hemocytes, respectively. Following LPS-stimulation, 79 genes were significantly upregulated and 265 genes were downregulated. The differentially expressed genes (DEGs) were related to multiple immune functional categories and pathways such as those of the cytoskeleton, Toll and Imd, apoptosis, MAP kinase (MAPK), inositol phosphate metabolism, phagosome, leucocyte endothelial migration, and gram-negative bacterial infection, among others. This study provides important information about the mechanisms of response to LPS, which is relevant for the understanding the HSCs’ immune response. Full article

The basal phylogeny of Chelicerata is one of the opaquest parts of the animal Tree of Life, defying resolution despite application of thousands of loci and millions of sites. At the forefront of the debate over chelicerate relationships is the monophyly of Arachnida, which has been refuted by most analyses of molecular sequence data. A number of phylogenomic datasets have suggested that Xiphosura (horseshoe crabs) are derived arachnids, refuting the traditional understanding of arachnid monophyly. This result is regarded as controversial, not least by paleontologists and morphologists, due to the widespread perception that arachnid monophyly is unambiguously supported by morphological data. Moreover, some molecular datasets have been able to recover arachnid monophyly, galvanizing the belief that any result that challenges arachnid monophyly is artefactual. Here, we explore the problems of distinguishing phylogenetic signal from noise through a series of in silico experiments, focusing on datasets that have recently supported arachnid monophyly. We assess the claim that filtering by saturation rate is a valid criterion for recovering Arachnida. We demonstrate that neither saturation rate, nor the ability to assemble a molecular phylogenetic dataset supporting a given outcome with maximal nodal support, is a guarantor of phylogenetic accuracy. Separately, we review empirical morphological phylogenetic datasets to examine characters supporting Arachnida and the downstream implication of a single colonization of terrestrial habitats. We show that morphological support of arachnid monophyly is contingent upon a small number of ambiguous or incorrectly coded characters, most of these tautologically linked to adaptation to terrestrial habitats. Full article