Microsatellite Loci of the Atlantic Horseshoe Crab (Limulus polyphemus) Reveal Inter-Localities Genetic Diversity in the Coastal Waters of the Eastern and Northern Yucatan Peninsula.

The American horseshoe crab (Limulus polyphemus) is an economically and ecologically important species, which is currently categorized as endangered in Mexico. L. polyphemus, one of four extant horseshoe crab species that constitute the class Merostomata, is distributed along the Atlantic coastline of the USA from Alabama to Maine and has another population on the coastline of Campeche, Yucatan, and Quintana Roo in the Yucatan Peninsula, Mexico. In the present study, we evaluated the genetic diversity and genetic structure of four separated localities along the coast of the Yucatan peninsula (Champoton, CH; Isla Arena, IA; Rio Lagartos, RL; and Holbox Island, HI), using nine microsatellite-type molecular markers for this species. The aim of this study is to obtain a baseline of the current level of genetic diversity, which would allow the monitoring of important changes over time. Multilocus analyses revealed moderate levels of genetic diversity (He, 0.5230 to 0.6389) and genetic structure within the whole study area (FST 0.025). The population from RL showed limited gene flows, differing significantly from the other sampling sites. The genetic information obtained in this study can support the implementation of management and conservation programs for this species in Mexico.

Microplastic transfer from the American horseshoe crab to shorebirds through consumption of horseshoe crab eggs in Jamaica Bay, NY.

Microplastic transfer between horseshoe crabs (Limulus polyphemus) and migratory shorebirds through consumption of crab eggs was examined in Jamaica Bay, New York. Fertilized and unfertilized crab eggs, shorebird fecal pellets, beach sand, and bay water were processed with a hydrogen peroxide solution to remove organic material, then stained with a Nile Red to identify microplastics using fluorescence microscopy. Microplastics were present in all samples and ranged from approximately26-1300 µm. Unfertilized and fertilized eggs contained significantly higher numbers of microplastic particles per gram than shorebird fecal pellets, beach sand, and bay water. The presence of microplastics in unfertilized egg samples indicates that microplastics undergo maternal transfer during oogenesis. We estimated that 1 g of horseshoe crab eggs could contain approximately 426 to 840 microplastic particles, suggesting that shorebirds feeding on this resource could be ingesting a substantial burden of microplastics during their migratory stopover, much of which appears to be retained by shorebirds, rather than being eliminated in their fecal pellets.

Differential Gene Expression Induced by Acute Exposure to Water Accommodated Fraction (WAF) and Chemically Enhanced WAF (CEWAF) of Light Crude Oil and Nokomis 3-F4 in Limulus polyphemus Larvae.

In 2018 we evaluated at 48 h and 96 h, the gene expression profile of larvae of Limulus polyphemus exposed to 10% and 100% of water-accommodated fraction (WAF) of light crude oil (API 35), and 10% and 100% of a chemically enhanced WAF (CEWAF) with the dispersant Nokomis 3-F4® in a static-acute (96 h) bioassay. Alkanes and PAHs concentrations were higher in CEWAF than in WAF stock solutions. Under the proved conditions, the expression profile of genes associated to detoxification processes (glutathione S-transferase and glutathione peroxidase), stress (heat shock protein), innate immunity (tumor necrosis factor receptor-associated factor 4 traf4), cell death (apoptosis inhibitor 5) and DNA repairing (E3 ubiquitin protein ligase), showed a deregulation at 48 h followed by an upregulation at 96 h, with exception of glutathione peroxidase, heat shock protein and innate immunity that remained low in CEWAF. In conclusion, by using genes that have been proposed as biomarkers to pollutants exposure, L. polyphemus larvae showed an early activation of genes related to the immune system, antioxidant, heat shock and NER.

First Evidence of Glyphosate in American Horseshoe Crab from the Yucatan Peninsula in Mexico.

Glyphosate is the most used herbicide in the world. Unfortunately, contamination of water bodies by this herbicide has been reported. A severe concern has been triggered given its detrimental impact on the environment and wildlife. The American horseshoe crab (Limulus polyphemus) is a benthic arthropod that inhabits the Yucatan Peninsula in Southeast Mexico. This study evaluates the glyphosate concentration in 34 recently dead specimens of L. polyphemus from four localities of the Ria Lagartos Biosphere Reserve in Yucatan, Mexico. The analysis was carried out using High-Performance Liquid Chromatography coupled with a Triple Quadrupole Mass Spectrometer. All the samples showed residues of glyphosate in the range from 0.08 to 2.38 ng g-1. These records constitute the first evidence of glyphosate bioaccumulation in this species. Although the scope might be limited, the results demonstrate a potentially prejudicial exposition of the marine biota to glyphosate-based herbicides, given its use in the region.

Environmental factors and occurrence of horseshoe crabs in the northcentral Gulf of Mexico.

This study provides regional-scale data on drivers of horseshoe crab (Limulus polyphemus) presence along the northcentral Gulf of Mexico coast and has implications for understanding habitat suitability for sparse horseshoe crab populations of conservation concern worldwide. To collect baseline data on the relationship between environmental factors and presence of horseshoe crabs, we surveyed four sites from the Fort Morgan peninsula of Mobile Bay, Alabama (AL) to Horn Island, Mississippi (MS). We documented number, size and sex of live animals, molts, and carcasses as metrics of horseshoe crab presence and demographics for two years. Data were compared to in situ and remotely sensed environmental attributes to assess environmental drivers of occurrence during the time of study. Overall, greater evidence of horseshoe crab presence was found at western sites (Petit Bois and Horn Islands) compared to eastern sites (Dauphin Island, Fort Morgan peninsula), mediated by a combination of distance from areas of high freshwater discharge and interannual variation in weather. Higher sex ratios also were found associated with higher occurrence, west of Mobile Bay. Land cover, particularly Bare Land and Estuarine Emergent Wetland classes that are common to western sites, was most predictive of live animal and to some extent carcass occurrence. Our findings suggest that small-scale variation in habitat quality can affect occurrence of horseshoe crabs in sparse populations where density is not a limiting factor. Data from molts and carcasses were informative to supplement live animal data and may be useful to enhance ecological assessment and support conservation and management in regions with sparse populations.

Antitumour peptide based on a protein derived from the horseshoe crab: CIGB-552 a promising candidate for cancer therapy.

Peptide-based cancer therapy has been of great interest due to the unique advantages of peptides, such as their low MW, the ability to specifically target tumour cells, easily available and low toxicity in normal tissues. Therefore, identifying and synthesizing novel peptides could provide a promising option for cancer patients. The antitumour second generation peptide, CIGB-552 has been developed as a candidate for cancer therapy. Proteomic and genomic studies have identified the intracellular protein COMMD1 as the specific target of CIGB-552. This peptide penetrates to the inside tumour cells to induce the proteasomal degradation of RelA, causing the termination of NF-κB signalling. The antitumour activity of CIGB-552 has been validated in vitro in different human cancer cell lines, as well as in vivo in syngeneic and xenograft tumour mouse models and in dogs with different types of cancers. The aim of this review is to present and discuss the experimental data obtained on the action of CIGB-552, including its mechanism of action and its therapeutic potential in human chronic diseases. This peptide is already in phase I clinical trials as antineoplastic drug and has also possible application for other inflammatory and metabolic conditions.

A comparison of pyrogen detection tests in the quality control of meningococcal conjugate vaccines: The applicability of the Monocyte Activation Test.

The meningococcal C conjugate vaccine (MenCC) is an interesting model with which to test the efficacy of the Monocyte Activation Test (MAT) as an alternative method of pyrogen testing in the quality control of vaccines. The MenCC that has been produced by Bio-Manguinhos in Brazil is in the final development stage, and, as recommended in the guidelines for MenCC production, its pyrogen content must be determined by using the Limulus Amoebocyte Lysate (LAL) assay and the Rabbit Pyrogen Test (RPT). This represents an ideal opportunity to compare LAL and RPT data with data obtained by using a MAT system with cryopreserved whole blood and IL-6/IL-1ß as marker readouts. In order to assess the compatibility of the MAT with MenCC, endotoxin and non-endotoxin pyrogen content was quantified by using MenCC samples spiked with lipopolysaccharide (LPS), lipoteichoic acid or zymosan standards. The presence of the aluminium-based adjuvant interfered with the MAT, increasing the readout of IL-1ß in LPS-spiked MenCC batches. This infringed the product-specific validation criteria of the test, and led to IL-6 being chosen as the more suitable marker readout. No pyrogenic contaminants were identified in the MenCC batches tested, demonstrating consistency among the different systems (MAT, RPT and the LAL assay). In conclusion, the introduction of the MAT during MenCC development could contribute to the elimination of animal tests post-licensing, ensuring human protection based on an effective non-animal based method of quality control.

Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL).

Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods.

Population structure of the red mangrove crab, Goniopsis cruentata (Decapoda: Grapsidae) under different fishery impacts: Implications for resource management / Estructura de la población del cangrejo rojo de manglar, Goniopsis cruentata (Decapoda: Grapsidae) bajo diferentes impactos de pesca: Implicaciones para la gestión de los recursos

The red mangrove crab, Goniopsis cruentata, influences the recruitment and composition of plant species in the mangrove ecosystem and it is an important fishery resource. Nevertheless, no current management and conservation plans are available for this species for the Brazilian coast. This investigation evaluated the population structure and reproductive biology in populations of G. cruentata under contrasting fishery pressures. The sampling program was carried out in two mangroves, Vaza-Barris and Sergipe River, from January through December 2011. Crabs from both mangroves were randomly collected by a professional fisherman during daytime low tide periods, using a fishing rod baited with pieces of a locally abundant gastropod, Pugilina morio, during 20min/area (catch per unit effort). Monthly measurements of air, sediment surface layer and water temperatures were obtained with a digital thermometer and salinity with an optical refractometer. Both crab populations were compared concerning their abundance, body size, sex ratio, size at onset of sexual maturity and fecundity (FI). Abiotic factors (air, water and mud temperature; and salinity) showed no significant differences between sampling localities. A total of 4 370 crabs were sampled, 2 829 from the Sergipe River and 1 541 from the Vaza-Barris River. The abundance and body size of crabs were compared between mangroves, and statistically significant differences were found. The sex ratio for both populations differed from the expected 1:1 ratio, and a significant deviation in favor of juvenile males was obtained, while adults showed a bias toward females. The estimated size at onset of sexual maturity for both sexes was similar in both populations. However, the populations differed significantly in the number and volume of eggs: a higher FI was obtained in females from the Sergipe River, while a higher egg volume was observed in females from the Vaza-Barris River mangrove. These results indicated a tendency to decrease the body size, the abundance of crabs and the reproductive potential of the species, with higher fishing intensities, reinforcing the need to develop a management plan as well as to establish conservation units for G. cruentata in the Northeastern Brazilian coast. Habitat loss in conjunction with long-term overfishing can have irreversible consequences, which can impact not only the populations of commercially exploited crabs, but the dynamics of virtually the entire mangrove ecosystem.

El cangrejo rojo Goniopsis cruentata influye en el reclutamiento y la composición de especies vegetales del ecosistema manglar y es un importante recurso pesquero. Sin embargo hasta la fecha, no se han desarrollado planes de manejo y conservación para esta especie en Brasil. La presente investigación evaluó la estructura poblacional y la biología reproductiva de las poblaciones de G. cruentata expuestas a diferentes presiones de pesca. El muestreo se realizó en dos manglares, Río Sergipe y Río Vaza-Barris entre enero y diciembre 2011. En ambos manglares, los animales fueron recolectados de día durante la marea baja por un pescador profesional, utilizando una caña de pescar con carnada del gasterópodo Pugilina morio, durante 20min/área (captura por unidad de esfuerzo, CPUE). Mensualmente con un termómetro digital se registró la temperatura del aire, agua y capa superficial del sedimento, mientras que la salinidad fue cuantificada utilizando un refractómetro óptico. Las poblaciones fueron comparadas en su abundancia, tamaño corporal, proporción de sexos, talla de primera madurez sexual y fecundidad. Los factores abióticos(temperatura del aire, agua y sedimento y la salinidad) no mostraron diferencias significativas entre ambos sitios de muestreo. Se recolectaron un total de 4 370 cangrejos; 2 829 en el río Sergipe y 1 541 en Vaza-Barris. Se registraron diferencias significativas entre los manglares con respecto a la abundancia y el tamaño corporal de los animales. La proporción de sexos de ambas poblaciones difirió de la relación 1:1 esperada y se obtuvo una desviación significativa en favor de machos jóvenes, mientras que los adultos mostraron una tendencia hacia las hembras. A su vez, las poblaciones difirieron significativamente en el número de huevos, con un mayor IF para las hembras del manglar del río Sergipe. El volumen de los huevos también mostró diferencias significativas entre ambas poblaciones. Estos resultados indican que la alta intensidad de pesca podría estar afectando dichos parámetros, lo que refuerza la necesidad de desarrollar un plan de manejo y la creación de unidades de conservación para G. cruentata en la costa noreste de Brasil. La pérdida del hábitat junto con la sobrepesca a largo plazo, pueden tener consecuencias irreversibles, que afectan no sólo a las poblaciones de cangrejos de importancia comercial, sino también la dinámica del ecosistema de manglar.

How pH, Temperature, and Time of Incubation Affect False-Positive Responses and Uncertainty of the LAL Gel-Clot Test.

The limulus amebocyte lysate (LAL) test is the simplest and most widely used procedure for detection of endotoxin in parenteral drugs. The LAL test demands optimal pH, ionic strength, temperature, and time of incubation. Slight changes in these parameters may increase the frequency of false-positive responses and the estimated uncertainty of the LAL test. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. LAL tests were performed in nominal conditions (37 °C, 60 min, and pH 7) and in different conditions of temperature (36 °C and 38 °C), time of incubation (58 and 62 min), and pH (6 and 8). Slight differences in pH increase the frequency of false-positive responses 5-fold (relative risk 5.0), resulting in an estimated of uncertainty 7.6%. Temperature and time of incubation affect the LAL test less, showing relative risks of 1.5 and 1.0, respectively. Estimated uncertainties in 36 °C or 38 °C temperatures and 58 or 62 min of incubation were found to be 2.0% and 1.0%, respectively. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses. LAY ABSTRACT: The limulus amebocyte lysate (LAL) gel-clot test is a simple test for detection of endotoxin from Gram-negative bacteria. The test is based on a gel formation when a certain amount of endotoxin is present; it is a pass/fail test. The LAL test requires optimal pH, ionic strength, temperature, and time of incubation. Slight difference in these parameters may increase the frequency of false-positive responses. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. We find that slight differences in pH increase the frequency of false-positive responses 5-fold. Temperature and time of incubation affect the LAL test less. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses.

A portable test system for determination of bacterial endotoxins in 18F-FDG, 99mTc, and lyophilized reagents for labeling with 99mTc.

UNLABELLED: A rapid quantitative kinetic chromogenic test in an automated portable test system has been developed for in-process and end-product determination of bacterial endotoxins in water using the Limulus amebocyte lysate. The aim of this work was to validate the method for (18)F-FDG, (99m)Tc, and the lyophilized reagents methylene diphosphonic acid (MDP) and pyrophosphate for labeling with (99m)Tc radiopharmaceuticals with no interfering factors. METHODS: Experiments were performed on 3 consecutive batches of (18)F-FDG, (99m)Tc, MDP, and pyrophosphate produced at the Nuclear Energy and Research Institute of São Paulo, Brazil, using a portable test system. The maximum valid dilution (=500) was calculated to establish the extent of dilution to avoid interfering test conditions. RESULTS: Better results were obtained above a 1:5 dilution factor for (18)F-FDG and (99m)Tc, 1:20 for MDP, and 1:100 for pyrophosphate. The requirements of the test were satisfied (R ≤ 0.980, recovery of product positive control between 50% and 200%, and coefficient variation of samples < 25%), and the endotoxin concentration was lower than the lowest concentration of the standard curve (0.05 endotoxin unit mL(-1)) and therefore less than the established limit in pharmacopoeias. CONCLUSION: The portable test system is a rapid, simple, and accurate technique using the quantitative kinetic chromogenic method for bacterial endotoxin determination. For this reason, the test is practical for radiopharmaceutical uses and tends to be the method of choice for the pyrogen test. For (18)F-FDG, (99m)Tc, MDP, and pyrophosphate, the validation was successfully performed.

A novel fusion protein-based vaccine comprising a cell penetrating and immunostimulatory peptide linked to human papillomavirus (HPV) type 16 E7 antigen generates potent immunologic and anti-tumor responses in mice.

The ultimate success of cancer vaccination is dependent upon the generation of tumor-specific CTLs. In this study, we designed and evaluated a novel fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32-51 of the Limulus polyphemus protein (LALF(32-51)) linked to human papillomavirus (HPV) 16 E7 antigen (LALF(32-51)-E7). We demonstrated that LALF(32-51) penetrates the cell membrane and delivers E7 into cells. In a preclinical model of HPV16-induced cervical carcinoma we showed that vaccination with adjuvant-free LALF(32-51)-E7 fusion protein significantly improves the presentation of E7-derived peptides to T-cells in vitro and induces suppression of tumor growth.

Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals.

Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent.

A Limulus antilipopolysaccharide factor-derived peptide exhibits a new immunological activity with potential applicability in infectious diseases.

Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of the Limulus antilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF(31-52) peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-gamma) and IFN-alpha. Analysis of the effect of LALF(31-52) on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF(31-52) protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-alpha levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.

Lipophorin as a lipid carrier in the hemolymph of the horseshoe crab, Limulus polyphemus.

A high-density lipoprotein (HDL) was obtained from the hemolymph of Limulus polyphemus in yields generally less than 30 micrograms/ml by ultracentrifugal flotation. SDS-PAGE revealed two apolipoproteins with masses similar to those of apolipophorins (apoLp-I, 265 +/- 14 kDa; apoLp-II, 89 +/- 6 kDa). Lipid composition was different from both insect lipophorin and crustacean HDL, and showed less diacylglycerols than triacylglycerols (3.8% and 36.2% of total lipids, respectively). Since Limulus polyphemus is closely related to precambrian chelicerates, our results confirm that lipophorin was present early in the evolution of arthropods.

Lipophorin as a lipid carrier in the hemolymph of the horseshoe crab, Limulus polyphemus

A high-density lipoprotein (HDL) was obtained from the hemolymph of Limulus polyphemus in yields generally less than 30 micrograms/ml by ultracentrifugal flotation. SDS-PAGE revealed two apolipoproteins with masses similar to those of apolipophorins (apoLp-I, 265 +/- 14 kDa; apoLp-II, 89 +/- 6 kDa). Lipid composition was different from both insect lipophorin and crustacean HDL, and showed less diacylglycerols than triacylglycerols (3.8 and 36.2 of total lipids, respectively). Since Limulus polyphemus is closely related to precambrian chelicerates, our results confirm that lipophorin was present early in the evolution of arthropods.

Determinación de los pirógenos provenientes de endotoxinas gramnegativas con el empleo del método del lisato de amebocitos del Limulus polyphemus (LAL) / Determination of pyrogens from gramnegative endotoxins with use of the method of limulus polyphemus amebocyte lysate (LAL)

Se informa que el Limulus polyphemus o cangrejo herradura es un invertebrado marino que vive en las costas del Golfo de México. Se expresa que su hemolinfa ha sido extraída para la obtención de un lisato de amebocitos que tiene la propiedad de gelificar cuando se pone en presencia de endotoxinas bacterianas. Se indica que dicha hemolinfa se ha aprovechado para la detección de las endotoxinas bacterianas en soluciones para el uso parenteral. Se decide que debido a las dificultades que se presentan con el animal que se emplea en el ensayo oficial, entre otras, se debe estudiar la posibilidad de implantar el método del lisato de amebocitos del Limulus polyphemus (LAL) para detectar las endotoxinas gramnegativas en nuestras producciones y comparar su efectividad frente al método tradicional. Se muestra en el presente trabajo la utilidad del montaje del LAL y su reproducibilidad, así como la factibilidad y la efectividad cuando se compara con el ensayo oficial