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.

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.

Effect of Benzophenone-3 to Acetylcholinesterase and Antioxidant System in Zebrafish (Danio rerio) Embryos.

Benzophenone-3 (BP-3) is one of the most used UV filters. The present study aimed to evaluate the toxic effects of BP-3 during embryo stages of zebrafish four hours post-fertilization (4hpf). Embryos were exposed to 0, 1, and 10 µg L-1 of BP-3 for 72 h. We investigated biochemical and molecular biomarkers of neurotoxicity (AChE) and the antioxidant system (gene expression of catalase, CAT, superoxide dismutase, SOD, glutathione peroxidase, GPX, the concentration of total glutathione, GSH, and lipid hydroperoxides, LPO). Results indicated that the acute exposure to BP-3 in zebrafish embryos did not show significant differences in survival, hatching rate, or antioxidant system biomarkers. In contrast, there were significant differences associated with AChE gene expression and activity.

Comparative physicochemical analysis of chitin quality of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus.

Recently, chitin biopolymer has received much attention as a wide variety of biomedical application for this and its derivatives have been reported, in fact, the study of non-conventional species as alternative sources of them compounds has taken particular interest. Here, we present a comparative physicochemical survey of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus: the prosoma and the opisthosoma, collected in Yucatán, Mexico. The characterization included CHNSO analysis, FTIR, TGA, DSC, XRD, and SEM. The CHNSO analysis revealed that C is present in the highest proportion (∼45 %) and that chemical composition did not show significant differences (P < 0.05) between the two tagmata. FTIR spectra of two tagmata presented a wide characteristic band of the chitin between 3600 and 3000 cm-1, confirming the presence of this biopolymer in the exoskeleton studied. TGA and DTGA profiles resulted very similar for both tagmata being the residual mass at 650 °C of around 30 % for both samples; these values were associated to the presence of minerals. SEM micrographs showed a porous matrix with infinite large number of irregularly shaped particles. Results show that both tagmata are made up of chitin, and they seem to have a high mineral content.

Humoral antibody response of the tilapia Oreochromis niloticus against Cichlidogyrus spp. (Monogenea).

The humoral immune response of the tilapia Oreochromis niloticus was evaluated using a direct ELISA. Serum was tested from fish infected with Cichlidogyrus spp. (Monogenea) and from fish injected intraperitoneally with the Cichlidogyrus spp. antigenic extract, i.e., 150 microl of the Cichlidogyrus spp. saline extract diluted in Freund's complete adjuvant (FCA) (1:1) were inoculated intraperitoneally at day 0, followed by 2 dosages of 50 microl of the same Cichlidogyrus spp. saline extract diluted in Freund's incomplete adjuvant (FIA) (1:1) at weeks 2 and 4, respectively. The humoral response was also evaluated by the double immunodiffusion test (DID) and by serum protein and total immunoglobulin (Ig) determinations. The IgM OD values in the hyperimmune fish were significantly higher than in the infected and uninfected fish groups. In the DID test, a precipitation (antigen-antibody) band was observed between the Cichlidogyrus spp. saline extract and hyperimmune sera, but not with the other groups. Increases in serum protein concentration and total Igs were observed in the immunized fish at weeks 2 and 10 postinjection. Results from this study suggest that tilapia is capable of producing an induced humoral immune response against an antigenic extract of Cichlidogyrus spp.