Sargassum spp. Ethanolic Extract Elicits Toxic Responses and Malformations in Zebrafish (Danio rerio) Embryos.

The amount of Sargassum spp. arriving in the Caribbean Sea has increased steadily in the last few years, producing a profound environmental impact on the ecological dynamics of the coasts of the Yucatan Peninsula. We characterized the toxicological effects of an ethanolic extract of Sargassum spp. on zebrafish (Danio rerio) embryos (ZFEs) in a 96-h static bioassay using T1 (0.01 mg/L), T2 (0.1 mg/L), T3 (1 mg/L), T4 (10 mg/L), T5 (25 mg/L), T6 (50 mg/L), T7 (75 mg/L), T8 (100 mg/L), T9 (200 mg/L), and T10 (400 mg/L). In this extract, we detected 74 compounds by gas chromatography-mass spectrometry (GC-MS), of which hexadecanoic acid methyl ester, and 2-pentanone 4-hydroxy-4-methyl, were the most abundant. In ZFEs, a median lethal concentration of 251 mg/L was estimated. Exposed embryos exhibited extensive morphological changes, including edema in the yolk sac, scoliosis, and loss of pigmentation, as well as malformations of the head, tail, and eyes. By integrating these abnormalities using the Integrated Biological Response (IBRv2) and General Morphological Score (GMS) indices, we were able to determine that ZFEs exposed to 200 mg/L (T9) exhibited the most pronounced biological response in comparison with the other groups. In the comparative transcriptomic analysis, 66 genes were upregulated, and 246 genes were downregulated in the group exposed to 200 mg/L compared with the control group. In the upregulated genes, we identified several gene ontology-enriched terms, such as response to xenobiotic stimuli, cellular response to chemical stimulus, transcriptional regulation, pigment metabolic process, erythrocyte differentiation and embryonic hemopoiesis, extracellular matrix organization, and chondrocyte differentiation involved in endochondral bone morphogenesis, among others. In the down-regulated genes, we found many genes associated with nervous system processes, sensory and visual perception, response to abiotic stimulus, and the nucleoside phosphate biosynthetic process. The probable connections among the morphological changes observed in the transcriptome are thoroughly discussed. Our findings suggest that Sargassum spp. exposure can induce a wide negative impact on zebrafish embryos. Environ Toxicol Chem 2024;43:1075-1089. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Integrative description of changes occurring on zebrafish embryos exposed to water-soluble crude oil components and its mixture with a chemical surfactant.

The effects of crude oil spills are an ongoing problem for wildlife and human health in both marine and freshwater aquatic environments. Bioassays of model organisms are a convenient way to assess the potential risks of the substances involved in oil spills. Zebrafish embryos (ZFE) are a useful to reach a fast and detailed description of the toxicity of the pollutants, including both the components of the crude oil itself and substances that are commonly used for crude oil spill mitigation (e.g. surfactants). Here, we evaluated the survival rate, as well as histological, morphological, and proteomic changes in ZFE exposed to Water Accumulated Fraction (WAF) of light crude oil and in mixture with Dioctyl Sulfosuccinate Sodium (DOSS, e.g. CEWAF: Chemically Enhanced WAF), a surfactant that is frequently used in chemical dispersant formulations. Furthermore, we compared the hydrocarbon concentration of WAF and CEWAF of the sublethal dilution. In histological, morphological, and gene expression variables, the ZFE exposed to WAF showed less changes than those exposed to CEWAF. Proteomic changes were more dramatic in ZFE exposed to WAF, with important alterations in spliceosomal and ribosomal proteins, as well as proteins related to eye and retinal photoreceptor development and heart function. We also found that the concentration of high molecular weight hydrocarbons in water was slighly higher in presence of DOSS, but the low molecular weight hydrocarbons concentration was higher in WAF. These results provide an important starting point for identifying useful crude-oil exposure biomarkers in fish species.

The surfactant Dioctyl Sodium Sulfosuccinate (DOSS) exposure causes adverse effects in embryos and adults of zebrafish (Danio rerio).

Dioctyl Sodium Sulfosuccinate (DOSS, CAS 577-11-7) is a chemical emulsifying surfactant that is widely used in the food and the cosmetic industry, and it is also the major component of the crude oil chemical dispersant Corexit™. Despite of its wide use, the studies related to its negative effect have been evaluated mainly in marine environments showing that DOSS is highly bioactive, extremely low volatile, and potential to persist in the environment longer than other dispersant components. Up to date, there is no available information of DOSS concentration in freshwater environments, little is known about its downstream fate after excretion and its effect on freshwater organisms. The objective of this study was to evaluate the effect of DOSS at different concentrations in embryos and adults of zebrafish Danio rerio in an acute-static bioassays of 96 h. The median lethal concentration in embryos was 33.3 mg/L. Malformations started to be observed at 10 mg/L. In adults, the gene expression analysis in gill tissues showed a deregulation in genes associated with the antioxidant system and the nucleotide excision repair mechanism. Additionally, Micronuclei (DNA damage) in erythrocytes, and fat degeneration in liver, hypertrophy and hyperplasia in gills, and hyaline drops in kidney tissues were also observed. In conclusion, the concentrations of DOSS evaluated here would be of health relevance to fish based on morphological alterations in embryos and changes in the gene expression profile, DNA damage and tissue impairment in adults.

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.

Red Blood Cell Cytotoxicity Associated to Heavy Metals and Hydrocarbons Exposure in Flounder Fish from Two Regions of the Gulf of Mexico.

In this study, the genotoxic effect of contaminants was assessed through detection of DNA damage using the micronucleus (MNs) test in erythrocytes from 149 flounder fish collected in two regions of the Gulf of Mexico (GoM). The frequency of microcytes (MCs) was also evaluated in the same group of fish collected from the Perdido Foldbelt (PF) and the Yucatan Platform (YP). The MCs frequency was different among locations of the YP (p = 0.011), while MNs frequency varied among locations of PF (p = 0.024). MCs and MNs values correlated with heavy metals from fish muscle, fish species and localities. Mean number, prevalence, and intensity of MCs and MNs correlated with Al, PAHs, depth, and locality. MNs frequency showed a species-specific association (p = 0.004). MNs and MCs were associated with heavy metals and PAHs from fish muscle and sediments, and the MNs frequency was species dependent.

Gill and liver transcriptomic responses of Achirus lineatus (Neopterygii: Achiridae) exposed to water-accommodated fraction (WAF) of light crude oil reveal an onset of hypoxia-like condition.

Crude oil is one of the most widespread pollutants released into the marine environment, and native species have provided useful information about the effect of crude oil pollution in marine ecosystems. We consider that the lined sole Achirus lineatus can be a useful monitor of the effect of crude oil in the Gulf of Mexico (GoM) because this flounder species has a wide distribution along the GoM, and its response to oil components is relevant. The objective of this study was to compare the transcriptomic changes in liver and gill of adults lined sole fish (Achirus lineatus) exposed to a sublethal acute concentration of water-accommodated fraction (WAF) of light crude oil for 48 h. RNA-Seq was performed to assess the transcriptional changes in both organs. A total of 1073 differentially expressed genes (DEGs) were detected in gills; 662 (61.69%) were upregulated, and 411 (38.30%) were downregulated whereas in liver, 515 DEGs; 306 (59.42%) were upregulated, and 209 (40.58%) were downregulated. Xenobiotic metabolism and redox metabolism, along with DNA repair mechanisms, were activated. The induction of hypoxia-regulated genes and the generalized regulation of multiple signaling pathways support the hypothesis that WAF exposition causes a hypoxia-like condition.

Alterations in the Gut Microbiota of Zebrafish (Danio rerio) in Response to Water-Soluble Crude Oil Components and Its Mixture With a Chemical Dispersant.

Crude oil spills have caused substantial impacts to aquatic ecosystems. Chemical dispersants are used to palliate the impact of oil spillages, but their use is polemic due to their additional potential toxic effect when mixed with oil-derived components. In this work, we used a 16S-based metagenomic approach to analyze the changes of the gut microbiota of adult zebrafish (Danio rerio) exposed to the water accommodated fraction (WAF) of a light crude oil (35° API gravity), and the chemically enhanced WAF (CEWAF), prepared with Nokomis 3-F4® dispersant. After 96 h of exposure, WAF induced an increase in the alpha and beta diversity, altering the relative abundance of Vibrio, Flavobacterium, and Novosphingobium. In contrast, CEWAF only caused an increase in the beta diversity, and an enrichment of the genus Pseudomona. Both treatments diminished the abundances of Aeromonas, Cetobacterium, Coxiella, Dinghuibacter, and Paucibacter. Moreover, the co-occurrence network among genera was more complex in WAF than in CEWAF, indicating a greater bacterial interaction in response to WAF. Our results indicate that short-term exposure to WAF and CEWAF can induce a dysbiosis in the gut microbiota of D. rerio, but these changes are specific in each treatment.

Effects of chronic exposure to water accommodated fraction (WAF) of light crude oil on gut microbiota composition of the lined sole (Achirus lineatus).

Exposure of marine fish to hydrocarbon compounds from crude oil can cause physiological and ecological alterations that can result in several cytotoxic, genotoxic, and teratogenic damages. One consequence of this exposure is the dysbiosis of the gut microbiota, where the normal bacterial composition is modified. Herein, we assessed the effect of the exposure to water accommodated fraction (WAF) of a light crude oil into the gut microbiota of a native species, the lined sole Achirus lineatus, a benthonic fish widely distributed in the Gulf of Mexico (GoM). We performed a chronic bioassay using two WAF concentrations (5 and 10% v/v), collecting lined sole entire gastrointestinal tracts for microbiota analyses at two timepoints, 14 and 28 days. Changes in the gut microbiota composition were determined by high throughput amplicon sequencing of the gene 16S rRNA. Diversity analyses showed that WAF exposure produced similar changes in the microbiota composition at both concentrations. Metagenomic functional prediction showed that these alterations could result in a shift in the gut redox status, towards a more anoxygenic environment. Enrichment of bacteria capable of use hydrocarbon as carbon source seems to be fast regardless time of exposure or WAF concentrations. Our results suggest that chronic WAF exposure can cause a dysbiosis in this benthic native species from the GoM.

Toxicity evaluation and microbiota response of the lined sole Achirus lineatus (Chordata: Achiridae) exposed to the light petroleum water-accommodated fraction (WAF).

Exposure to contaminants might directly affect organisms and alter their associated microbiota. The objective of the present study was to determine the impact of the petroleum-water-accommodated fraction (WAF) from a light crude oil (API gravity 35) on a benthic fish species native from the Gulf of Mexico (GoM). Ten adults of Achirus lineatus (Linnaeus, 1758) were exposed to a sublethal WAF/water solution of 50% v/v for 48 hr. Multiple endpoints were measured including tissue damage, presence of polycyclic aromatic hydrocarbons (PAHs) metabolites in bile and gut microbiota analyses. Atrophy and fatty degeneration were observed in livers. Nodules and inflammation were detected in spleen, and structural disintegration and atrophy in the kidney. In gills hyperplasia, aneurysm, and gills lamellar fusion were observed. PAHs metabolites concentrations in bile were significantly higher in exposed organisms. Gut microbiome taxonomic analysis showed significant shifts in bacterial structure and composition following WAF exposure. Data indicate that exposure to WAF produced toxic effects in adults of A. lineatus, as evidenced by histological alterations and dysbiosis, which might represent an impairment to long-term subsistence of exposed aquatic organisms.

Integrating expert knowledge and ecological niche models to estimate Mexican primates' distribution.

Ecological niche modeling is used to estimate species distributions based on occurrence records and environmental variables, but it seldom includes explicit biotic or historical factors that are important in determining the distribution of species. Expert knowledge can provide additional valuable information regarding ecological or historical attributes of species, but the influence of integrating this information in the modeling process has been poorly explored. Here, we integrated expert knowledge in different stages of the niche modeling process to improve the representation of the actual geographic distributions of Mexican primates (Ateles geoffroyi, Alouatta pigra, and A. palliata mexicana). We designed an elicitation process to acquire information from experts and such information was integrated by an iterative process that consisted of reviews of input data by experts, production of ecological niche models (ENMs), and evaluation of model outputs to provide feedback. We built ENMs using the maximum entropy algorithm along with a dataset of occurrence records gathered from a public source and records provided by the experts. Models without expert knowledge were also built for comparison, and both models, with and without expert knowledge, were evaluated using four validation metrics that provide a measure of accuracy for presence-absence predictions (specificity, sensitivity, kappa, true skill statistic). Integrating expert knowledge to build ENMs produced better results for potential distributions than models without expert knowledge, but a much greater improvement in the transition from potential to realized geographic distributions by reducing overprediction, resulting in better representations of the actual geographic distributions of species. Furthermore, with the combination of niche models and expert knowledge we were able to identify an area of sympatry between A. palliata mexicana and A. pigra. We argue that the inclusion of expert knowledge at different stages in the construction of niche models in an explicit and systematic fashion is a recommended practice as it produces overall positive results for representing realized species distributions.

Genetic signature analysis of Perkinsus marinus in Mexico suggests possible translocation from the Atlantic Ocean to the Pacific coast of Mexico.

BACKGROUND: The protozoan Perkinsus marinus (Mackin, Owen & Collier) Levine, 1978 causes perkinsosis in the American oyster Crassostrea virginica Gmelin, 1791. This pathogen is present in cultured C. virginica from the Gulf of Mexico and has been reported recently in Saccostrea palmula (Carpenter, 1857), Crassostrea corteziensis (Hertlein, 1951) and Crassostrea gigas (Thunberg, 1793) from the Mexican Pacific coast. Transportation of fresh oysters for human consumption and repopulation could be implicated in the transmission and dissemination of this parasite across the Mexican Pacific coast. The aim of this study was two-fold. First, we evaluated the P. marinus infection parameters by PCR and RFTM (Ray's fluid thioglycollate medium) in C. virginica from four major lagoons (Términos Lagoon, Campeche; Carmen-Pajonal-Machona Lagoon complex, Tabasco; Mandinga Lagoon, Veracruz; and La Pesca Lagoon, Tamaulipas) from the Gulf of Mexico. Secondly, we used DNA sequence analyses of the ribosomal non-transcribed spacer (rNTS) region of P. marinus to determine the possible translocation of this species from the Gulf of Mexico to the Mexican Pacific coast. RESULTS: Perkinsus marinus prevalence by PCR was 57.7% (338 out of 586 oysters) and 38.2% (224 out of 586 oysters) by RFTM. The highest prevalence was observed in the Carmen-Pajonal-Machona Lagoon complex in the state of Tabasco (73% by PCR and 58% by RFTM) and the estimated weighted prevalence (WP) was less than 1.0 in the four lagoons. Ten unique rDNA-NTS sequences of P. marinus [termed herein the "P. marinus (Pm) haplotype"] were identified in the Gulf of Mexico sample. They shared 96-100% similarity with 18 rDNA-NTS sequences from the GenBank database which were derived from 16 Mexican Pacific coast infections and two sequences from the USA. The phylogenetic tree and the haplotype network showed that the P. marinus rDNA-NTS sequences from Mexico were distant from the rDNA-NTS sequences of P. marinus reported from the USA. The ten rDNA-NTS sequences described herein were restricted to specific locations displaying different geographical connections within the Gulf of Mexico; the Carmen-Pajonal-Machona Pm1 haplotype from the state of Tabasco shared a cluster with P. marinus isolates reported from the Mexican Pacific coast. CONCLUSIONS: The rDNA-NTS sequences of P. marinus from the state of Tabasco shared high similarity with the reference rDNA-NTS sequences from the Mexican Pacific coast. The high similarity suggests a transfer of oysters infected with P. marinus from the Mexican part of the Gulf of Mexico into the Mexican Pacific coast.

Panulirus argus virus 1 detected in oceanic postlarvae of Caribbean spiny lobster: implications for disease dispersal.

Panulirus argus virus 1 (PaV1), a pathogenic virus that specifically attacks Caribbean spiny lobsters Panulirus argus, was recently detected in newly settled postlarvae of P. argus. As PaV1 appears not to be vertically transmitted, infected postlarvae likely acquire PaV1 from the water, but whether this can occur in oceanic waters where the planktonic larvae (phyllosomata) metamorphose into nektonic postlarvae remains unknown. Late-stage phyllosomata and postlarvae of P. argus were collected at distances of 2 to 100 km from the Caribbean coast of Mexico in 2 oceanographic cruises. Most postlarvae were caught in the upper meter of water, usually along with masses of floating Sargassum algae. A PaV1-PCR assay was used to test 169 phyllosomata (stages VI-X) and 239 postlarvae. All phyllosomata tested negative, but 2 postlarvae, 1 from each cruise, tested positive for PaV1. These postlarvae were collected at 55 and 48 km offshore over depths of 850 and 1800 m, respectively, suggesting that postlarvae can acquire PaV1 in offshore waters. We hypothesize that floating Sargassum may be an environmental reservoir for PaV1. The PaV1 allele (460 pb) found in an infected postlarva was more closely related to PaV1 alleles found in lobsters from Puerto Rico than in lobsters from any other location (including Mexico), suggesting high gene flow and long-distance dispersal of PaV1, consistent with previous studies of high genetic connectivity across the Caribbean.

Noninvasive collection of fresh hairs from free-ranging howler monkeys for DNA extraction.

The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh-plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low-cost noninvasive technique for collecting fresh-plucked hairs used to obtain DNA samples from free-ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex-age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction-based microsatellite marker analysis. The technique proved successful for collecting fresh-plucked hairs of free-ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species.