Combining chemical, bioanalytical and predictive tools to assess persistence, seasonality, and sporadic releases of organic micropollutants within the urban water cycle.

Using a novel liquid chromatography-tandem mass spectrometry method with large volume direct injection and quantitation via isotope dilution, we evaluated the presence of 55 organic micropollutants in wastewater effluents, and locations within the Bow River and Elbow River watersheds in and around the city of Calgary, Alberta, Canada. In addition to establishing baseline micropollutant data for water utility operations, our study aimed to enhance our understanding of micropollutant behavior in the urban water cycle, assess the contributions of three wastewater treatment plants (WWTPs) to downstream receiving waters, explain the potential causes of total estrogenicity measured using the yeast-estrogen screen assay (YES), and prioritize a subset of substances for continuous monitoring. With data spanning 48 months and 95 river km, our results indicate the extensive persistence of metformin (antidiabetic), seasonality of N,N­diethyl-m-toluamide (DEET, insect repellant), O-desmethylvenlafaxine (antidepressant metabolite), and sulfamethoxazole (antibiotic) in source waters, and sporadic detections of a well-known perfluoroalkyl substance (PFOA). The seasonality of pharmaceuticals at the sentinel downstream monitoring site appeared to coincide with river dilution while that of DEET was likely attributable to peak usage during the warmer months. Steroidal estrogens were rarely detected in wastewater effluents although total estrogenicity via YES was evident, suggesting the presence of less potent but more abundant non-steroidal estrogens (e.g., flame retardants, bisphenols, and phthalates). A conservative mass balance analysis suggests that the largest WWTP (serving a population of >1 million) consistently contributed the highest load of micropollutants, with the exception of metformin, which appeared to be influenced by a smaller WWTP (serving 115,000) that operates a different activated sludge process. We consider metformin, sucralose, diclofenac, and venlafaxine as more effective conservative tracers of wastewater pollution due to their notably higher concentrations and persistence in the Bow River compared to carbamazepine and caffeine, respectively. Finally, hierarchical clustering revealed a close association between E. coli and caffeine, supporting the use of caffeine as an indicator of short-term, untreated anthropogenic inputs. Overall, this study yields valuable insights on the presence, behavior, and sources of organic micropollutants in the urban water cycle and identifies indicators of anthropogenic impacts that are useful for prioritizing future monitoring campaigns in Calgary and elsewhere.

First sex modification case in equine cloning.

Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic profiles, as observed in the analysis of 15-horse microsatellite marker panel, which confirmed they are indeed clones of the same animal. Cytogenetic analysis and fluorescent in situ hybridization using X and Y specific probes revealed a 63,X chromosome set in the female offspring, suggesting a spontaneous Y chromosome loss. The identity of the lost chromosome in the female was further confirmed through PCR by observing the presence of X-linked markers and absence of Y-linked markers. Moreover, cytogenetic and molecular profiles were analyzed in blood and skin samples to detect a possible mosaicism in the female, but results showed identical chromosomal constitutions. Although the cause of the spontaneous chromosome loss remains unknown, the possibility of equine sex reversal by SCNT holds great potential for the preservation of endangered species, development of novel breeding techniques, and sportive purposes.

Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer.

The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

Venlafaxine in Embryos Stimulates Neurogenesis and Disrupts Larval Behavior in Zebrafish.

Venlafaxine, a widely prescribed antidepressant, is a selective serotonin and norepinephrine reuptake inhibitor in humans, and this drug is prevalent in municipal wastewater effluents. While studies have shown that this drug affects juvenile fish behavior, little is known about the developmental impact on nontarget aquatic animals. We tested the hypothesis that venlafaxine deposition in the egg, mimicking maternal transfer of this antidepressant, disrupts developmental programming using zebrafish (Danio rerio) as a model. Embryos (1-4 cell stage) were microinjected with either 1 or 10 ng venlafaxine, which led to a rapid reduction (90%) of this drug in the embryo at hatch. There was a concomitant increase in the concentration of the major metabolite o-desmethylvenlafaxine during the same period. Embryonic exposure to venlafaxine accelerated early development, increased hatching rate and produced larger larvae at 5 days post fertilization. Also, there was an increase in neuronal birth in the hypothalamus, dorsal thalamus, posterior tuberculum, and the preoptic region, and this corresponded with a higher spatial expression of nrd4, a key marker of neurogenesis. The venlafaxine-exposed larvae were less active and covered shorter distance in a light and dark behavioral test compared to the controls. Overall, zygotic exposure to venlafaxine disrupts early development, including brain function, and compromises larval behavior, suggesting impact of this drug on developmental programming in zebrafish.

In vivo analysis of hyaloid vasculature morphogenesis in zebrafish: A role for the lens in maturation and maintenance of the hyaloid.

Two vascular networks nourish the embryonic eye as it develops - the hyaloid vasculature, located at the anterior of the eye between the retina and lens, and the choroidal vasculature, located at the posterior of the eye, surrounding the optic cup. Little is known about hyaloid development and morphogenesis, however. To begin to identify the morphogenetic underpinnings of hyaloid formation, we utilized in vivo time-lapse confocal imaging to characterize morphogenesis of the zebrafish hyaloid through 5 days post fertilization (dpf). Our data segregate hyaloid formation into three distinct morphogenetic stages: Stage I: arrival of hyaloid cells at the lens and formation of the hyaloid loop; Stage II: formation of a branched hyaloid network; Stage III: refinement of the hyaloid network. Utilizing fixed and dissected tissues, distinct Stage II and Stage III aspects of hyaloid formation were quantified over time. Combining in vivo imaging with microangiography, we demonstrate that the hyaloid system becomes fully enclosed by 5dpf. To begin to identify the molecular and cellular mechanisms underlying hyaloid morphogenesis, we identified a recessive mutation in the mab21l2 gene, and in a subset of mab21l2 mutants the lens does not form. Utilizing these "lens-less" mutants, we determined whether the lens was required for hyaloid morphogenesis. Our data demonstrate that the lens is not required for Stage I of hyaloid formation; however, Stages II and III of hyaloid formation are disrupted in the absence of a lens, supporting a role for the lens in hyaloid maturation and maintenance. Taken together, this study provides a foundation on which the cellular, molecular and embryologic mechanisms underlying hyaloid morphogenesis can be elucidated.