A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0.

The zebrafish model has emerged as a reference tool for phenotypic drug screening. An increasing number of molecules have been brought from bench to bedside thanks to zebrafish-based assays over the last decade. The high homology between the zebrafish and the human genomes facilitates the generation of zebrafish lines carrying loss-of-function mutations in disease-relevant genes; nonetheless, even using this alternative model, the establishment of isogenic mutant lines requires a long generation time and an elevated number of animals. In this study, we developed a zebrafish-based high-throughput platform for the generation of F0 knock-out (KO) models and the screening of neuroactive compounds. We show that the simultaneous inactivation of a reporter gene (tyrosinase) and a second gene of interest allows the phenotypic selection of F0 somatic mutants (crispants) carrying the highest rates of mutations in both loci. As a proof of principle, we targeted genes associated with neurodevelopmental disorders and we efficiently generated de facto F0 mutants in seven genes involved in childhood epilepsy. We employed a high-throughput multiparametric behavioral analysis to characterize the response of these KO models to an epileptogenic stimulus, making it possible to employ kinematic parameters to identify seizure-like events. The combination of these co-injection, screening and phenotyping methods allowed us to generate crispants recapitulating epilepsy features and to test the efficacy of compounds already during the first days post fertilization. Since the strategy can be applied to a wide range of indications, this study paves the ground for high-throughput drug discovery and promotes the use of zebrafish in personalized medicine and neurotoxicity assessment.

Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates.

The early identification of teratogens in humans and animals is mandatory for drug discovery and development. Zebrafish has emerged as an alternative model to traditional preclinical models for predicting teratogenicity and other potential chemical-induced toxicity hazards. To prove its predictivity, we exposed zebrafish embryos from 0 to 96 h post fertilization to a battery of 31 compounds classified as teratogens or non-teratogens in mammals. The teratogenicity score was based on the measurement of 16 phenotypical parameters, namely heart edema, pigmentation, body length, eye size, yolk size, yolk sac edema, otic vesicle defects, otoliths defects, body axis defects, developmental delay, tail bending, scoliosis, lateral fins absence, hatching ratio, lower jaw malformations and tissue necrosis. Among the 31 compounds, 20 were detected as teratogens and 11 as non-teratogens, resulting in 94.44 % sensitivity, 90.91 % specificity and 87.10 % accuracy compared to rodents. These percentages decreased slightly when referred to humans, with 87.50 % sensitivity, 81.82 % specificity and 74.19 % accuracy, but allowed an increase in the prediction levels reported by rodents for the same compounds. Positive compounds showed a high correlation among teratogenic parameters, pointing out at general developmental delay as major cause to explain the physiological/morphological malformations. A more detailed analysis based on deviations from main trends revealed potential specific modes of action for some compounds such as retinoic acid, DEAB, ochratoxin A, haloperidol, warfarin, valproic acid, acetaminophen, dasatinib, imatinib, dexamethasone, 6-aminonicotinamide and bisphenol A. The high degree of predictivity and the possibility of applying mechanistic approaches makes zebrafish a powerful model for screening teratogenicity.

How many came home? Evaluating ex situ conservation of green turtles in the Cayman Islands.

Ex situ management is an important conservation tool that allows the preservation of biological diversity outside natural habitats while supporting survival in the wild. Captive breeding followed by re-introduction is a possible approach for endangered species conservation and preservation of genetic variability. The Cayman Turtle Centre Ltd was established in 1968 to market green turtle (Chelonia mydas) meat and other products and replenish wild populations, thought to be locally extirpated, through captive breeding. We evaluated the effects of this re-introduction programmme using molecular markers (13 microsatellites, 800-bp D-loop and simple tandem repeat mitochondrial DNA sequences) from captive breeders (N = 257) and wild nesting females (N = 57) (sampling period: 2013-2015). We divided the captive breeders into three groups: founders (from the original stock), and then two subdivisions of F1 individuals corresponding to two different management strategies, cohort 1995 ("C1995") and multicohort F1 ("MCF1"). Loss of genetic variability and increased relatedness was observed in the captive stock over time. We found no significant differences in diversity among captive and wild groups, and similar or higher levels of haplotype variability when compared to other natural populations. Using parentage and sibship assignment, we determined that 90% of the wild individuals were related to the captive stock. Our results suggest a strong impact of the re-introduction programmme on the present recovery of the wild green turtle population nesting in the Cayman Islands. Moreover, genetic relatedness analyses of captive populations are necessary to improve future management actions to maintain genetic diversity in the long term and avoid inbreeding depression.

Population genomics of an endemic Mediterranean fish: differentiation by fine scale dispersal and adaptation.

The assessment of the genetic structuring of biodiversity is crucial for management and conservation. For species with large effective population sizes a low number of markers may fail to identify population structure. A solution of this shortcoming can be high-throughput sequencing that allows genotyping thousands of markers on a genome-wide approach while facilitating the detection of genetic structuring shaped by selection. We used Genotyping-by-Sequencing (GBS) on 176 individuals of the endemic East Atlantic peacock wrasse (Symphodus tinca), from 6 locations in the Adriatic and Ionian seas. We obtained a total of 4,155 polymorphic SNPs and we observed two strong barriers to gene flow. The first one differentiated Tremiti Islands, in the northwest, from all the other locations while the second one separated east and south-west localities. Outlier SNPs potentially under positive selection and neutral SNPs both showed similar patterns of structuring, although finer scale differentiation was unveiled with outlier loci. Our results reflect the complexity of population genetic structure and demonstrate that both habitat fragmentation and positive selection are on play. This complexity should be considered in biodiversity assessments of different taxa, including non-model yet ecologically relevant organisms.

Root biomechanics in Rhizophora mangle: anatomy, morphology and ecology of mangrove's flying buttresses.

BACKGROUND AND AIMS: Rhizophora species of mangroves have a conspicuous system of stilt-like roots (rhizophores) that grow from the main stem and resemble flying buttresses. As such, the development of rhizophores can be predicted to be important for the effective transmission of dynamic loads from the top of the tree to the ground, especially where the substrate is unstable, as is often the case in the habitats where Rhizophora species typically grow. This study tests the hypothesis that rhizophore architecture in R. mangle co-varies with their proximity to the main stem, and with stem size and crown position. METHODS: The allometry and wood mechanical properties of R. mangle (red mangrove) trees growing in a mangrove basin forest within a coastal lagoon in Mexico were compared with those of coexisting, non-buttressed mangrove trees of Avicennia germinans. The anatomy of rhizophores was related to mechanical stress due to crown orientation (static load) and to prevailing winds (dynamic load) at the study site. KEY RESULTS: Rhizophores buttressed between 10 and 33 % of tree height. There were significant and direct scaling relationships between the number, height and length of rhizophores vs. basal area, tree height and crown area. Wood mechanical resistance was significantly higher in the buttressed R. mangle (modulus of elasticity, MOE = 18·1 ± 2 GPa) than in A. germinans (MOE = 12·1 ± 0·5 GPa). Slenderness ratios (total height/stem diameter) were higher in R. mangle, but there were no interspecies differences in critical buckling height. When in proximity to the main stem, rhizophores had a lower length/height ratio, higher eccentricity and higher xylem/bark and pith proportions. However, there were no directional trends with regard to prevailing winds or tree leaning. CONCLUSIONS: In comparison with A. germinans, a tree species with wide girth and flare at the base, R. mangle supports a thinner stem of higher mechanical resistance that is stabilized by rhizophores resembling flying buttresses. This provides a unique strategy to increase tree slenderness and height in the typically unstable substrate on which the trees grow, at a site that is subject to frequent storms.

Tough adults, frail babies: an analysis of stress sensitivity across early life-history stages of widely introduced marine invertebrates.

All ontogenetic stages of a life cycle are exposed to environmental conditions so that population persistence depends on the performance of both adults and offspring. Most studies analysing the influence of abiotic conditions on species performance have focussed on adults, while studies covering early life-history stages remain rare. We investigated the responses of early stages of two widely introduced ascidians, Styela plicata and Microcosmus squamiger, to different abiotic conditions. Stressors mimicked conditions in the habitats where both species can be found in their distributional ranges and responses were related to the selection potential of their populations by analysing their genetic diversity. Four developmental stages (egg fertilisation, larval development, settlement, metamorphosis) were studied after exposure to high temperature (30°C), low salinities (26 and 22‰) and high copper concentrations (25, 50 and 100 µg/L). Although most stressors effectively led to failure of complete development (fertilisation through metamorphosis), fertilisation and larval development were the most sensitive stages. All the studied stressors affected the development of both species, though responses differed with stage and stressor. S. plicata was overall more resistant to copper, and some stages of M. squamiger to low salinities. No relationship was found between parental genetic composition and responses to stressors. We conclude that successful development can be prevented at several life-history stages, and therefore, it is essential to consider multiple stages when assessing species' abilities to tolerate stress. Moreover, we found that early development of these species cannot be completed under conditions prevailing where adults live. These populations must therefore recruit from elsewhere or reproduce during temporal windows of more benign conditions. Alternatively, novel strategies or behaviours that increase overall reproductive success might be responsible for ensuring population survival.

Tracking invasion histories in the sea: facing complex scenarios using multilocus data.

In recent years, new analytical tools have allowed researchers to extract historical information contained in molecular data, which has fundamentally transformed our understanding of processes ruling biological invasions. However, the use of these new analytical tools has been largely restricted to studies of terrestrial organisms despite the growing recognition that the sea contains ecosystems that are amongst the most heavily affected by biological invasions, and that marine invasion histories are often remarkably complex. Here, we studied the routes of invasion and colonisation histories of an invasive marine invertebrate Microcosmus squamiger (Ascidiacea) using microsatellite loci, mitochondrial DNA sequence data and 11 worldwide populations. Discriminant analysis of principal components, clustering methods and approximate bayesian computation (ABC) methods showed that the most likely source of the introduced populations was a single admixture event that involved populations from two genetically differentiated ancestral regions--the western and eastern coasts of Australia. The ABC analyses revealed that colonisation of the introduced range of M. squamiger consisted of a series of non-independent introductions along the coastlines of Africa, North America and Europe. Furthermore, we inferred that the sequence of colonisation across continents was in line with historical taxonomic records--first the Mediterranean Sea and South Africa from an unsampled ancestral population, followed by sequential introductions in California and, more recently, the NE Atlantic Ocean. We revealed the most likely invasion history for world populations of M. squamiger, which is broadly characterized by the presence of multiple ancestral sources and non-independent introductions within the introduced range. The results presented here illustrate the complexity of marine invasion routes and identify a cause-effect relationship between human-mediated transport and the success of widespread marine non-indigenous species, which benefit from stepping-stone invasions and admixture processes involving different sources for the spread and expansion of their range.

Migration of As, Hg, Pb, and Zn in arroyo sediments from a semiarid coastal system influenced by the abandoned gold mining district at El Triunfo, Baja California Sur, Mexico.

Extensive waste deposits (tailings) and ash from the ignition oven of the abandoned gold mine of mining district El Triunfo (MD-ET) in Baja California Sur, Mexico have released trace elements into the sediments of the Hondo-Las Gallinas-El Carrizal arroyo, which connects to the Pacific Ocean through an evaporitic basin. Migration of these elements through the arroyo is mainly caused by winds or tropical hurricanes that occur sporadically during the summer and cause the otherwise dry arroyo to overflow. To evaluate the concentration and distribution of the elements As, Hg, Pb, and Zn along the 48 km arroyo, surface sediments were collected from 26 sites, ranging from close to the MD-ET to the mouth of the arroyo at the Pacific Ocean. Concentrations in tailings and ash were for As 8890 and 505 000 mg kg(-1); for Hg 0.336 and 54.9 mg kg(-1); for Pb 92,700 and 19,300 mg kg(-1); and for Zn 49,600 and 1380 mg kg(-1). The average of the Normalized Enrichment Factor (Av-NEF) in surface sediments, calculated using background levels, indicates that the sediments are severely contaminated with As and Zn (Av-NEF = 22), Pb (Av-NEF = 24) and with a moderate contamination of Hg (Av-NEF = 7.5). The anthropogenic influence of those elements is reflected in the arroyo sediments as far as 18 km away from the MD-ET, whereas the samples closest to the discharge into the Pacific Ocean show a natural to moderate enrichment for As and Zn and low or no enrichment for Hg and Pb. A principal components analysis identified four principal components that explained 90% of the total variance. Factor 1 was characterized by a high positive contribution of the anthropogenic source elements, especially As, Pb, and Zn (37%), whereas Factor 2 was strongly correlated with the oxy-hydroxides of Fe and Mn (27%). Factor 3 was correlated with Li (16%) and Factor 4 with Al (10%), which indicates more than one source of lithogenic composition, though they played a minor role in the distribution of the elements.

The effect of ultrasonic treatmen on some functional properties of egg white

This work studied the effect of treatment with a high frequency ultrasound bath (40 kHz and a power of 80 W.) on the pH, foaming capacity and stability of the foam of the white of hens eggs (sample 1/120 hours post laying and sample 2/24 hours post laying). Exposure time was at varied intervals of 0, 5, 10 and 15 mins at 20°C. The samples were stored in refrigeration at 4°C for 3 days. The comparative study of the data was carried out using variance analysis(ANOVA) and surface response analysis (RSA); where it was found that the pH did not show any significant changes(P<0.05) due to the effect of the treatment and remained during storage. On the other hand, the results showed that treatment with ultrasound for ten minutes increased the foaming capacity by 15.84 and 13.07 percent in samples 1 and 2, respectively. However, the stability of the foam in both samples was seen to diminish by 13.89 and 8.50 percent.

En este trabajo se estudió el efecto del tratamiento con un baño de ultrasonido de alta frecuencia (40 kHz y una potencia de 80 W) durante 0; 5; 10 y 15 min. / 20°C, sobre el pH, capacidad espumante y estabilidad de la espuma de la clara de huevos de gallina (muestra 1/ 120 horas pos-postura y muestra 2/ 24 horas post-postura). Las muestras fueron almacenadas en refrigeración a 4°C durante 3 días. El estudio comparativo de los datos se realizó por análisis de varianza (ANOVA) y superficie de respuesta (RSA); donde se encontró que en el pH no presentó cambios significativos (P<0,05) por efecto del tratamiento y de igual manera se mantuvo durante el tiempo de almacenamiento. Por otro lado se observó que el tratamiento con ultrasonido durante 10 mins aumentó la capacidad espumante en un 15,84 y 13,07 por ciento en las muestras 1 y 2, respectivamente. Sin embargo, la estabilidad de la espuma de estas muestras se vio disminuida en un 13,89 y 8,50 por ciento.