Differential blood cells associated with parasitism in the wild puffer fish Lagocephalus laevigatus (Tetraodontiformes) of the Campeche Coast, southern Mexico.

In aquaculture conditions, severe parasitic infections cause negative impacts on fish health and economic losses. The parasite load has been associated with anemia, which reduces the number of erythrocytes in fish. Therefore, the evaluation of hematological parameters as a feasible tool for diagnosing and monitoring fish health allows us to determine the indirect effect of parasites on the health status of fish. Our aim was to evaluate changes in the blood cell parameters of Lagocephalus laevigatus associated with parasitism. A total of 99 puffer fish were collected from the coast of Seybaplaya, Campeche. Each fish had 20 µl of peripheral blood drawn, and blood smears were performed in triplicate. The smears were stained with Giemsa stain, and a quantitative analysis of blood cells (erythrocytes, leukocytes, and monocytes) was obtained with an optical microscope at 100 ×. The parasites recovered from each fish were fixed and identified, and the infection parameters were calculated. Through generalized additive model analysis (GAMLSS), we observed that the infection intensity of puffer fish influenced changes in hematological parameters, principally in erythrocytes, neutrophils, thrombocytes, the total fish length, and the condition factor of the fish. In conclusion, this is the first study that provides baseline data on the hematological parameter variations in uninfected and infected L. laevigatus, the tropical wild puffer fish, as well as the possible effects on fish health. It is necessary to establish reference hematological patterns in wild populations for diagnosis and timely management with emphasis on aquaculture fish.

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.

DNA detection of Gyrodactylus spp. in skin mucus of Nile tilapia Oreochromis niloticus.

Monogeneans Gyrodactylus von Nordmann 1832, cause outbreaks of gyrodactylosis in aquaculture settings worldwide. Detection of Gyrodactylus spp. is based on the morphological identification of isolated parasites after fish necropsy. Contributing to the diagnosis of gyrodactylosis, in this study, a non-destructive PCR assay was standardized; the PCR was first performed using genomic DNA of Gyrodactylus spp. isolated from the surface of the Nile tilapia Oreochromis niloticus (Linnaeus 1758), and subsequently tested with mucus samples of infected and uninfected Nile tilapia fish. The primers (Ekgyro1) were designed from the ribosomal Internal Transcriber Spacer (ITS) RNA region (ITS1, 5.8S and ITS2 rRNA gene) of Gyrodactylus cichlidarum Paperna 1968. The positive control group included the DNA of 30 monogeneans Gyrodactylus spp. The heterologous control group included 75 monogeneans Cichlidogyrus Paperna 1960, 75 protozoans Ichthyophthirius multifiliis Fouquet 1876 and 75 Trichodina Ehrenberg 1830. PCR products of each parasite and from the external mucus samples (described as P and M respectively), were sequenced. The average DNA concentration of the ectoparasites was of 13.5 ng/µl. The PCR test had an analytical sensitivity of 0.0039 ng µl-1 of DNA of Gyrodactylus spp. No cross-reactions were observed with the heterologous group. The sensitivity and specificity of the PCR test were of 100% either with genomic DNA or with DNA from mucus samples. Six DNA consensus sequences with sizes ranging from 568 bp to 571 bp were obtained and the BLAST analysis matched with DNA sequences of G. cichlidarum.

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.

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.