Morphological Alterations in the Early Developmental Stages of Zebrafish (Danio rerio; Hamilton 1822) Induced by Exposure to Polystyrene Microparticles.

Microplastics (MPs) are emerging pollutants of widespread concern in aquatic environments. The aim of this study was to evaluate the negative impact of pristine MPs of polystyrene of 100 µm on embryo and larvae of Danio rerio exposed to three environmentally relevant concentrations of polystyrene (3.84 × 10- 6, 3.84 × 10- 7, and 3.84 × 10- 8 g/mL). The exposure effect was evaluated through the general morphology score, biometrics, and integrated biomarker response version 2 index. No mortality was observed but the anatomical structure of fishes was affected showing pigmentation deficiency and alterations in the head region as the main affected endpoints. The general morphology score and the integrated biomarker response values were highly sensitive to address the effect of the three concentrations of MPs used here. Our results provide solid evidence of the negative impact of 100 µm pristine polystyrene MPs exposure on early stages of zebrafish.

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.

A first glimpse into the transcriptomic changes induced by the PaV1 infection in the gut of Caribbean spiny lobsters, Panulirus argus (Latreille, 1804) (Decapoda: Achelata: Palinuridae).

The Caribbean spiny lobster, Panulirus argus (Latreille, 1804) supports important fisheries in the Caribbean region. This species is affected by a deadly virus, Panulirus argus Virus 1 (PaV1), the only known pathogenic virus for this species. As infection progresses, the effects of PaV1 on its host become systemic, with far reaching impacts on the host's physiology, including structural injuries to its gastrointestinal organs, such as the hepatopancreas and the gut. This last one becomes highly compromised in the last stages of infection. Since the gut is a key organ for the physiological stability of lobsters, we compared the transcriptomic changes in the gut of juvenile individuals of Panulirus argus naturally infected with PaV1. In the RNA-Seq analysis, we obtained a total of 485 × 106 raw reads. After cleaning, reads were de novo assembled into 68,842 transcripts and 50,257 unigenes. The length of unigenes ranged from 201 bp to 28,717 bp, with a N50 length of 2079, and a GC content of 40.61%. In the differential gene expression analysis, we identified a total of 3405 non redundant differential transcripts, of which 1920 were up-regulated and 1485 were down-regulated. We found alterations in transcripts encoding for proteins involved in transcriptional regulation, splicing, postraductional regulation, protein signaling, transmembrane transport, cytoskeletal regulation, and proteolysis, among others. This is the first insight into the transcriptomic regulation of PaV1-P. argus interaction. The information generated can help to unravel the molecular mechanisms that may intervene in the gut during PaV1 infection.

Comparative Transcriptomes of the Body Wall of Wild and Farmed Sea Cucumber Isostichopus badionotus.

Overfishing of sea cucumber Isostichopus badionotus from Yucatan has led to a major population decline. They are being captured as an alternative to traditional species despite a paucity of information about their health-promoting properties. The transcriptome of the body wall of wild and farmed I. badionotus has now been studied for the first time by an RNA-Seq approach. The functional profile of wild I. badionotus was comparable with data in the literature for other regularly captured species. In contrast, the metabolism of first generation farmed I. badionotus was impaired. This had multiple possible causes including a sub-optimal growth environment and impaired nutrient utilization. Several key metabolic pathways that are important in effective handling and accretion of nutrients and energy, or clearance of harmful cellular metabolites, were disrupted or dysregulated. For instance, collagen mRNAs were greatly reduced and deposition of collagen proteins impaired. Wild I. badionotus is, therefore, a suitable alternative to other widely used species but, at present, the potential of farmed I. badionotus is unclear. The environmental or nutritional factors responsible for their impaired function in culture remain unknown, but the present data gives useful pointers to the underlying problems associated with their aquaculture.

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.

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.

Physiological and immunological characterization of Caribbean spiny lobsters Panulirus argus naturally infected with Panulirus argus Virus 1 (PaV1).

The present study compares 13 physiological and immunological variables between a group of healthy Panulirus argus lobsters and a group of lobsters naturally infected with Panulirus argus Virus 1 (PaV1). Viral infection was determined through histopathology and PCR. Ten of the 13 variables differed significantly between the 2 groups. Using these variables, a principal component analysis yielded 2 separate clusters: one corresponding to the healthy group and the other corresponding to the infected group. In particular, infected lobsters exhibited significantly lower levels of osmotic pressure, total hemocyte counts, plasmatic proteins, and total phenoloxidase (PO) activity in plasma, as well as significantly higher levels of cholesterol and acylglycerides. These features are consistent with metabolic wasting, hyperlipidemia, and presumed immune suppression. Infection with PaV1 appears to increase the susceptibility of lobsters to some other opportunistic pathogens, as 61.1% of infected lobsters presented infestations of ciliate epibionts (Epystilis and Zoothamniun) in the gill chamber compared with 11.5% lobsters in the healthy group. Infected lobsters also showed significantly higher levels of total PO activity in degranulated hemocytes and trypsin inhibitor activity, potentially indicating activation of immune response by the PO system during the systemic infection with PaV1.

Non-lethal detection of DNA from Cichlidogyrus spp. (Monogenea, Ancyrocephalinae) in gill mucus of the Nile tilapia Oreochromis niloticus.

Infection of Nile tilapia Oreochromis niloticus by monogeneans of the genus Cichlidogyrus is harmful. Currently, diagnosis of this infection is based on invasive techniques and the identification of isolated parasites by their morphology. To facilitate diagnosis, we have developed a non-lethal polymerase chain reaction (PCR) test for detection of Cichlidogyrus spp. DNA in the gill mucus of O. niloticus, using 5 pairs of specific primers based on Cichlidogyrus sclerosus 28S rRNA (Cicly 1 to Cicly 5) which generate fragments of approximately 188, 180, 150, 159 and 189 bp, respectively. PCR specificity was tested using genomic DNA extracted individually from 175 isolated Cichlidogyrus spp., 75 Gyrodactylus cichlidarum and 75 endopararasitic Enterogyrus spp., as well as from 75 protozoans Trichodina spp. The Cicly primers were used to detect Cichlidogyrus spp. DNA in mucus from the gills of 23 Nile tilapia confirmed to be infected with the parasite. Negative controls consisted of 45 uninfected Nile tilapia. The limit of sensitivity of the assay was 1.2 ng of purified parasite DNA. The Cicly primers did not amplify DNA from the mucus of non-infected Nile tilapia, G. cichlidarum, Trichodina spp. or Enterogyrus spp. In all cases, the sensitivity and specificity of the test were 100%. The sequences of all the amplified fragments showed a high similarity to that of the 28S rRNA region of C. sclerosus (93 to 100% identical to GenBank Accession No. DQ157660.1). We provide evidence for a safe and non-invasive DNA-based diagnostic method for the presence of Cichlidogyrus in the gill mucus of O. niloticus.

Lessons from a study in a rural community from southern Mexico: risk factors associated to transmission and reinfection of gastrointestinal parasites after albendazole treatment.

PURPOSE: To determine the prevalence of gastrointestinal parasites and evaluate the effect of a single dose of treatment with albendazole in a sentinel group from a rural community in southern Mexico. METHODS: Stool samples were collected from 1456 individuals aged ≥1 year during consecutive days, and examined for helminth infection using the modified Stoll dilution method. Additionally, 104 individuals were treated with a single dose of albendazole and evaluated over 21 weeks to assess reinfection. Questionnaires were administered to obtain individual and household-level data pertaining to behavior, demography, and socioeconomic status. Risk factors for reinfection after albendazole administration were determined using multiple logistic regression analyses. RESULTS: The prevalence of Ascaris lumbricoides was 73.9% (95% confidence interval [CI] = 71.56%-76.14%). Albendazole was 100% effective, but eggs began to be detected by 9-12 weeks posttreatment, increasing to 100% after 21 weeks. Logistic regression analysis revealed that all individuals from this study had a probability of reinfection of 1.65× each week after treatment. The prevalence of Trichuris trichiura was 57.2% (95% CI = 54.62%-59.77%) and chemotherapy was 34.7% effective. The prevalence for other minor gastrointestinal parasites ranged from 0.2% to 29.7%. CONCLUSION: This was a comprehensive study on gastrointestinal parasites in a rural community from southern Mexico and, to the best of the authors' knowledge, is the first time that the effect of albendazole has been evaluated for a period of over 21 weeks following its administration. Risk factors associated with parasite transmission were linked to poverty and lack of hygiene, such as, defecating in open places, living customs (drinking piped water and/or from a well), and absence of knowledge about transmission life cycle of the parasites. Studies of this kind should be linked to health education and improvement of access to clean water and adequate sanitation to consolidate morbidity control and enhance sustainability.

Detection of Panulirus argus Virus 1 (PaV1) in exported frozen tails of subadult-adult Caribbean spiny lobsters Panulirus argus.

The Caribbean spiny lobster Panulirus argus is a valuable fishing resource and the trade in frozen lobster tails is an important industry. However, the presence of the pathogenic virus Panulirus argus Virus 1 (PaV1), which causes systemic infection in P. argus and is particularly lethal to juvenile individuals, has not been previously examined in imported/exported lobster products. We used PCR assays to determine the presence of PaV1 in abdominal muscle tissue of 22 frozen P. argus tails exported from Belize to Mexico. Based on their size, the tails belonged to subadult-adult lobsters. Using specific primers targeted for PaV1 resulted in 11 tails showing a specific 499 bp band. The sequence of positive amplified fragments showed a high similarity to PaV1 (95% identity with GenBank accession no. EF206313.1). Although the pathogenicity of PaV1 was not evaluated in the present study, our results provide the first evidence of PaV1 in frozen lobster tails exported in the seafood industry as well as the first molecular evidence of PaV1 in adult lobsters.