Mercury Concentration, DNA Methylation, and Mitochondrial DNA Damage in Olive Ridley Sea Turtle Embryos With Schistosomus Reflexus Syndrome.

Schistosomus reflexus syndrome (SR) is a rare and lethal congenital malformation that has been reported in the olive ridley sea turtle (Lepidochelys olivacea) in Mexico. Although the etiology remains unclear, it is presumed to be genetic. Since embryonic development in sea turtles largely depends on environmental conditions, we investigated whether sea turtle total mercury content participates in the etiology of SR. Given that several toxins are known to affect both DNA methylation and/or mitochondrial DNA (mtDNA) copy number, we also probed for associations of these parameters to SR and mercury exposure. We measured the levels of each variable in malformed olive ridley sea turtle embryos (either with SR or other non-SR malformations) and embryos without malformations. Malformed embryos (with or without SR) showed higher mercury concentrations compared to normal embryos, while only embryos with SR showed higher levels of methylation compared to embryos without malformations and those with other malformations. Furthermore, we uncovered a positive correlation between mercury concentrations and DNA methylation in SR embryos. With respect to mtDNA copy number, no differences were detected across experimental groups. Because of sample size limitations, this study is an initial attempt to understand the association of environmental toxins (such as mercury) and epigenetic alterations (DNA methylation) in the etiology of SR in sea turtles.

Methylation status of the putative Pax6 promoter in olive ridley sea turtle embryos with eye defects: An initial approach.

Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5'-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.

First approximation to congenital malformation rates in embryos and hatchlings of sea turtles.

BACKGROUND: Congenital malformations in sea turtles have been considered sporadical. Research carried out in the Mexican Pacific revealed high levels of congenital malformations in the olive ridley, but little or no information is available for other species. We present results from analyses of external congenital malformations in olive ridley, green, and hawskbill sea turtles from Mexican rookeries on the Pacific coast and Gulf of Mexico. METHODS: We examined 150 green and hawksbill nests and 209 olive ridley nests during the 2010 and 2012 nesting seasons, respectively. Olive ridley eggs were transferred to a hatchery and incubated in styrofoam boxes. Nests from the other two species were left in situ. Number of eggs, live and dead hatchlings, and eggs with or without embryonic development were registered. Malformation frequency was evaluated with indices of prevalence and severity. RESULTS: Mortality levels, prevalence and severity were higher in olive ridley than in hawksbill and green sea turtles. Sixty-three types of congenital malformations were observed in embryos, and dead or live hatchlings. Of these, 38 are new reports; 35 for wild sea turtles, three for vertebrates. Thirty-one types were found in hawksbill, 23 in green, and 59 in olive ridley. The head region showed a higher number of malformation types. Malformation levels in the olive ridley were higher than previously reported. CONCLUSION: Olive ridleys seem more prone to the occurrence of congenital malformations than the other two species. Whether the observed malformation levels are normal or represent a health problem cannot be currently ascertained without long-term assessments.

Malformaciones en embriones y neonatos de tortuga golfina (Lepidochelys olivacea) en Nuevo Vallarta, Nayarit, México / Malformations in embryos and neonates of Olive Ridley sea turtle (Lepidochelys olivacea) in Nuevo Vallarta, Nayarit, Mexico

With the aim to know the types of malformations in Olive Ridley sea turtle (Lepidochelys olivacea) embryos and neonates, and determine their prevalence and intensity indexes and %² in Nuevo Vallarta Nayarit, 100 nests were surveyed. From each nest, the total number of eggs and the number of embryos and neonates with any kind of abnormalities were registered. In 21 types of malformations located on seven anatomical regions, three new types were registered: anterior flippers amelia, bifurcated anterior flippers and rinodimo. The carapace presented significantly higher frequency of abnormalities (41.7%), followed by mandibles and general malformations (17.6% each one). The most frecuent type of malformation was the compressed carapace (26.9%), followed by albinism (14.8%) and xiphosis (13.2%). Prevalence (50% on nests and 1.8% on the organisms) and intensity (2.78 malformed organisms per nest and 1.57 malformations per organism) were high medium, compared to previous studies. Sea turtles from Bahía de Banderas may be exposed to anthropogenic contaminants, but is necessary to do long term studies to corroborate this possible relationship.

Con el propósito de conocer las malformaciones en embriones y neonatos de tortuga golfina (Lepidochelys olivacea) en Nuevo Vallarta, Nayarit, México, se revisaron 100 nidos de dichos animales para registrar el número total de huevos, de embriones y de neonatos con algún tipo de malformación; los datos se analizaron con índices de prevalencia e intensidad y pruebas de Ji cuadrada. En 21 tipos de malformaciones localizadas en siete regiones anatómicas, se registraron tres nuevos tipos: bifurcación de aletas anteriores, biamelia anterior y rinodimo. La región corporal con mayor frecuencia de malformaciones fue el caparazón (41.7%), seguida por las mandíbulas y las malformaciones generales (17.6% cada una). El tipo de malformación más frecuente fue el caparazón comprimido (26.9%), seguido de albinismo (14.8%) y quifosis (13.2%). La prevalencia (50% de los nidos y 1.8% de los organismos) y la intensidad (2.78 malformaciones/nido y 1.57 malformaciones/organismo) resultaron medianamente altas, en comparación con estudios previos. Las tortugas marinas de Bahía de Banderas pueden estar expuestas a contaminantes antropogénicos, pero aún falta hacer estudios a largo plazo para corroborar esta posible relación.