Search Results (17)

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

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

Gram-negative bacterial endotoxins can cause pathophysiological effects such as high fever when introduced into the bloodstream. Therefore, endotoxin testing is necessary when producing injectable pharmaceuticals. The pharmaceutical industry has widely used Limulus amebocyte lysate (LAL) to certify product quality. However, ethical concerns have been raised and the increasing scarcity of Limulus polyphemus necessitates the development of novel testing techniques. Recombinant factor C (rFC) was developed using genetic engineering techniques. The aim of this study was to investigate the validity of rFC testing and compare it with the LAL method. The specificity, linearity, accuracy, precision, and robustness of the rFC assay were evaluated. After validation, the rFC assay was found to be suitable for endotoxin detection. We compared the accuracy of the rFC and LAL assays using reference standard endotoxin. The rFC assay was as accurate as the LAL assay. We also compared the two assays using biopharmaceuticals. Greater interference occurred in some samples when the rFC assay was used than when the LAL assay was used. However, the rFC assay overcame the interference when the samples were diluted. Overall, we suggest that rFC can be applied to test biopharmaceuticals. 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

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

The Nymphalidae family of cosmopolitan butterflies (Lepidoptera) comprises approximately 7200 species found on all continents and in all habitats. However, debate persists regarding the phylogenetic relationships within this family. In this study, we assembled and annotated eight mitogenomes of Nymphalidae, constituting the first report of complete mitogenomes for this family. Comparative analysis of 105 mitochondrial genomes revealed that the gene compositions and orders were identical to the ancestral insect mitogenome, except for Callerebia polyphemus trnV being before trnL and Limenitis homeyeri having two trnL genes. The results regarding length variation, AT bias, and codon usage were consistent with previous reports on butterfly mitogenomes. Our analysis indicated that the subfamilies Limenitinae, Nymphalinae, Apaturinae, Satyrinae, Charaxinae, Heliconiinae, and Danainae are monophyletic, while the subfamily the subfamily Cyrestinae is polyphyletic. Danainae is the base of the phylogenetic tree. At the tribe level, Euthaliini in Limenitinae; Melitaeini and Kallimini in Nymphalinae; Pseudergolini in Cyrestinae; Mycalesini, Coenonymphini, Ypthimini, Satyrini, and Melanitini in Satyrinae; and Charaxini in Charaxinae are regarded as monophyletic groups. However, the tribe Lethini in Satyrinae is paraphyletic, while the tribes Limenitini and Neptini in Limenitinae, Nymphalini and Hypolimni in Nymphalinae, and Danaini and Euploeini in Danainae are polyphyletic. This study is the first to report the gene features and phylogenetic relationships of the Nymphalidae family based on mitogenome analysis, providing a foundation for future studies of population genetics and phylogenetic relationships within this family. Full article

Erythrocyte sedimentation rate (ESR) is a hematological test that can detect inflammatory activity within the body. Although not specific for any particular disease, ESR is often used as a screening “sickness indicator” due to its reliability and low cost. The Westergren method is a manual ESR technique commonly used but requires special graduated pipettes and over 1mL of whole blood, precluding its use in smaller patients where limited sample volumes can be obtained. A modified micro-ESR technique has been described using hematocrit capillary tubes but is used less commonly. ESR has been reported to be a useful inflammatory indicator in gopher tortoises (Gopherus polyphemus) and box turtles (Terrapene spp.) but not in Florida cottonmouth snakes (Agkistrodon conanti). Having an inexpensive screening test for inflammation can help guide medical decisions within conservation efforts of imperiled species. This study evaluated the correlation between these two ESR methodologies in threatened eastern indigo snakes (Drymarchon couperi, EIS) and found a very strong correlation (r_s = 0.897), without constant or proportional biases and a reference interval of 0 (90% CI -1-1)–9 mm/h (90% CI 8-11) was defined. Additionally, a significant difference was found between healthy EIS and EIS in mid-ecdysis (p = 0.006) and EIS with gastric cryptosporidiosis (p = 0.006), indicating ESR as a useful inflammatory indicator in EIS. Full article

Gopher Tortoise (Gopherus polyphemus) burrows support diverse commensal invertebrate communities that may be of special conservation interest. We investigated the impact of red imported fire ants (Solenopsis invicta) on the invertebrate burrow community at 10 study sites in southern Mississippi, sampling burrows (1998–2000) before and after bait treatments to reduce fire ant populations. We sampled invertebrates using an ant bait attractant for ants and burrow vacuums for the broader invertebrate community and calculated fire ant abundance, invertebrate abundance, species richness, and species diversity. Fire ant abundance in gopher tortoise burrows was reduced by >98% in treated sites. There was a positive treatment effect on invertebrate abundance, diversity, and species richness from burrow vacuum sampling which was not observed in ant sampling from burrow baits. Management of fire ants around burrows may benefit both threatened gopher tortoises by reducing potential fire ant predation on hatchlings, as well as the diverse burrow invertebrate community. Fire-ant management may also benefit other species utilizing tortoise burrows, such as the endangered Dusky Gopher Frog and Schaus swallowtail butterfly. This has implications for more effective biodiversity conservation via targeted control of the invasive fire ant at gopher tortoise burrows. Full article

Poland faces two great challenges in the field of environment and atmosphere protection: improving air quality, especially by reducing particulate matter (PM) emissions, and reducing relatively high greenhouse gas emissions. The aim of this research was to investigate how the fuel and technological transformations in the power, road transport, and household and tertiary sectors aimed at reducing carbon dioxide (CO₂) emissions in Poland would affect air quality, human health, and the associated external costs. The study was conducted for 2050 while considering 2015 as the base year. Ambient PM2.5 (particles with a diameter of less than 2.5 µm) concentration was used as a proxy air quality indicator. The analysis was based on decarbonization scenarios developed within the REFLEX Project (Analysis of the European energy system under the aspects of flexibility and technological progress). The three scenarios of the REFLEX Project focused on the reduction of CO₂ emissions up to 2050 from various sectors, mainly by the means of fuel and technological switches. This also led to the changes in the emission levels of pollutants that directly affect air quality, which were calculated with the use of fuel- and technology-specific emission factors. Next, for each emission scenario, ambient concentrations of PM2.5 and others pollutants were calculated with the use of the Polyphemus—an Eulerian-type air quality modelling system. Subsequently, the health impact of population exposed to air pollution and associated external costs were calculated using the πESA (Platform for Integrated Energy System Analysis) platform. The health impacts considered were the number of years of life lost, restricted activity days, and number of chronic bronchitis cases. The results showed that the largest reductions in both greenhouse gas and PM emissions—and consequently improvements of air quality resulting in a decrease of negative impacts on human health and a decrease of external costs—can be achieved by the transformation of heat production in the household and tertiary sector. The results also showed that the decrease in PM2.5 emissions envisaged in the analyzed scenarios in 2050 will lead to a reduction in the number of lost years of life by about 35 thousand and an avoidance of external costs by EUR 2.4 billion. Full article

A full-length cDNA sequence encoding a GnRH receptor was cloned from the pleuropedal ganglion of the Pacific abalone, Haliotis discus hannai. The cloned sequence is 1499-bp in length encoding a protein of 460 amino acid residues, with a molecular mass of 52.22 kDa and an isoelectric point (pI) of 9.57. The architecture of HdhGnRH-R gene exhibited key features of G protein-coupled receptors (GPCRs), including seven membrane spanning domains, putative N-linked glycosylation motifs, and phosphorylation sites of serine and threonine residues. It shared 63%, 52%, and 30% sequence identities with Octopus vulgaris, Limulus polyphemus, and Mizuhopecten yessoensis GnRH-R II sequences, respectively. Phylogenetic analysis indicated that HdhGnRH-R gene was clustered with GnRH-R II of O. vulgaris and O. bimaculoides. qPCR assay demonstrated that the mRNA expression level of this receptor was significantly higher in the pleuropedal ganglion than that in any other examined tissue. Transcriptional activities of this gene in gonadal tissues were significantly higher in the ripening stage. The mRNA expression of this gene was significantly higher in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000 °C). In situ hybridization revealed that HdhGnRH-R mRNA was expressed in neurosecretory cells of pleuropedal ganglion. Our results suggest that HdhGnRH-R gene synthesized in the neural ganglia might be involved in the control of gonadal maturation and gametogenesis of H. discus hannai. This is the first report of GnRH-R in H. discus hannai and the results may contribute to further studies of GPCRs evolution or may useful for the development of aquaculture method of this abalone species. Full article

Understanding the relationship between heavy metal and selenium levels in biota and their foods is important, but often difficult to determine because animals eat a variety of organisms. Yet such information is critical to managing species populations, ecological integrity, and risk to receptors (including humans) from consumption of certain prey. We examine levels of cadmium, lead, mercury, and selenium in biota from Delaware Bay (New Jersey, USA) to begin construction of a “springtime” food web that focuses on shorebirds. Horseshoe crab (Limulus polyphemus) eggs are one of the key components at the base of the food web, and crab spawning in spring provides a food resource supporting a massive stopover of shorebirds. Fish and other biota also forage on the crab eggs, and a complex food web leads directly to top-level predators such as bluefish (Pomatomus saltatrix) and striped bass (Morone saxatilis), both of which are consumed by egrets, eagles, ospreys (Pandion haliaetus), and humans. Metal levels in tissues were generally similar in algae, invertebrates, and small fish, and these were similar to those in blood of shorebirds (but not feathers). There was a significant direct relationship between the levels of metals in eggs of horseshoe crabs and mean metal levels in the blood of four species of shorebirds. Metal levels in shorebird feathers were higher than those in blood (except for selenium), reflecting sequestration of metals in feathers during their formation. Levels in feathers of laughing gulls (Leucophaeus atricilla) were similar to those in feathers of shorebirds (except for selenium). Selenium bears special mention as levels were significantly higher in the blood of all shorebird species than in other species in the food web, and were similar to levels in their feathers. Levels of metals in bluefish and striped bass were similar or higher than those found in the blood of shorebirds (except for selenium). The mean levels of cadmium, lead, and mercury in the blood and feathers of shorebirds were below any effect levels, but selenium levels in the blood and feathers of shorebirds were higher than the sublethal effect levels for birds. This is a cause for concern, and warrants further examination. Full article

Variation in DNA methylation patterns among genes, individuals, and populations appears to be highly variable among taxa, but our understanding of the functional significance of this variation is still incomplete. We here present the first whole genome bisulfite sequencing of a chelicerate species, the social spider Stegodyphus dumicola. We show that DNA methylation occurs mainly in CpG context and is concentrated in genes. This is a pattern also documented in other invertebrates. We present RNA sequence data to investigate the role of DNA methylation in gene regulation and show that, within individuals, methylated genes are more expressed than genes that are not methylated and that methylated genes are more stably expressed across individuals than unmethylated genes. Although no causal association is shown, this lends support for the implication of DNA CpG methylation in regulating gene expression in invertebrates. Differential DNA methylation between populations showed a small but significant correlation with differential gene expression. This is consistent with a possible role of DNA methylation in local adaptation. Based on indirect inference of the presence and pattern of DNA methylation in chelicerate species whose genomes have been sequenced, we performed a comparative phylogenetic analysis. We found strong evidence for exon DNA methylation in the horseshoe crab Limulus polyphemus and in all spider and scorpion species, while most Parasitiformes and Acariformes species seem to have lost DNA methylation. Full article

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. Full article

Recent scholarship addressing access to Homer’s epics during the Italian Renaissance has illuminated the unique importance of visual narratives for the dissemination and interpretation of material associated with the Trojan War and its heroes. This article looks at early fifteenth-century images deriving from the Odyssey that were painted for marriage chests (cassoni) in the popular Florentine workshop of Apollonio di Giovanni. Focusing on Apollonio’s subnarrative of Odysseus’ clash with the Cyclops Polyphemus (the Cyclopeia), I argue that Apollonio showcased this archetypal tale of a failed guest–host relationship to explore contemporary anxieties associated with marriage, an institution that figured prominently in the political and economic ambitions of fifteenth-century patriarchal families. Full article