In ovo manipulation of Nile crocodile embryos: egg windowing and potential dental research applications.

Crocodilians exhibit continuous tooth replacement (i.e., polyphyodonty) and have been identified as suitable models for tooth regeneration research due to the similarity in dental cavity and tooth anatomy between these creatures and humans. Various studies reporting in ovo bird embryo manipulation exist, but such reports for reptiles are virtually non-existent. Egg windowing enables direct access to oviparous vertebrate embryos and is therefore an important component of in ovo embryo manipulation experiments. The aim of the present study was to window Nile crocodile, Crocodylus niloticus eggs and assess the potential of direct manipulations, targeting the pharyngeal region where the maxilla and mandible originates. Crocodylus niloticus eggs were successfully windowed, and a limited number of individuals survived the entire gestation period. The 1st trimester of gestation was the most sensitive, and 96.78% of the mortalities occurred within this period. Our data indicate the suitable window for embryo manipulation targeting the mandibular arch and maxillary process, without a risk of damaging the chorioallantoic membrane (CAM) (which may be fatal), was between day six and eight after laying for embryos incubated at 31°C. This data will be of use for future embryo-based experiments related to jaw and tooth development in crocodiles as well as human tooth regeneration research.

Exposure Impacts of Environmentally Relevant Concentrations of a Glufosinate Ammonium Herbicide Formulation on Larval Development and Thyroid Histology of Xenopus laevis.

Thyroid hormones play critical roles in body growth and development as well as reproduction. They also influence the activities of a wider variety of tissues and biological functions, such as osmoregulation, metabolism, and especially metamorphosis in organisms, such as frogs. These complex activities of thyroid hormones are prone to disruption by agricultural pesticides, often leading to modulation of growth and the reproductive system in particular. These substances include Glufosinate ammonium, Glyphosates, Imazapyr, Penoxsulam, and Diquat dibromide among other herbicides. In this study, the standardized Xenopus Metamorphosis Assay protocol was used to assess the potential thyroid-modulatory properties of the Glufosinate ammonium Basta formulation, at relevant environmental concentrations (0.05 mg/L, 0.15 mg/L, and 0.25 mg/L) for 21 days. The results showed that this formulation only reduced the hind-limb length among the morphological endpoints. Histological evaluation showed that the mean thyroid gland area and the mean thyroidal follicle epithelium height were significantly increased following 0.15 and 0.25 mg/L exposures. The present study confirmed that this Basta formulation interacts with the thyroid axis and therefore potentially pose health hazard to amphibian in particular and potentially metamorphic aquatic vertebrates. Furthermore, the result is a signal of inherent potential thyroid disrupting activities that must be further investigated and characterised in some of the aquatic herbicide formulations to safeguard the aquatic biodiversity.

DNA Methylation and Demethylation Are Regulated by Functional DNA Methyltransferases and DnTET Enzymes in Diuraphis noxia.

Aphids are economically important insect pests of crops worldwide. Despite resistant varieties being available, resistance is continuously challenged and eventually broken down, posing a threat to food security. In the current study, the epigenome of two related Russian wheat aphid (Diuraphis noxia, Kurdjumov) biotypes (i.e., SA1 and SAM) that differ in virulence was investigated to elucidate its role in virulence in this species. Whole genome bisulfite sequencing covered a total of 6,846,597,083 cytosine bases for SA1 and 7,397,965,699 cytosine bases for SAM, respectively, of which a total of 70,861,462 bases (SA1) and 74, 073,939 bases (SAM) were methylated, representing 1.126 ± 0.321% (SA1) and 1.105 ± 0.295% (SAM) methylation in their genomes. The sequence reads were analyzed for contexts of DNA methylation and the results revealed that RWA has methylation in all contexts (CpG, CHG and CHH), with the majority of methylation within the CpG context (± 5.19%), while the other contexts show much lower levels of methylation (CHG - ± 0.27%; CHH - ± 0.34%). The top strand was slightly (0.02%) more methylated than the bottom strand. Of the 35,493 genes that mapped, we also analyzed the contexts of methylation of each of these and found that the CpG methylation was much higher in genic regions than in intergenic regions. The CHG and CHH levels did not differ between genic and intergenic regions. The exonic regions of genes were more methylated (±0.56%) than the intronic regions. We also measured the 5mC and 5hmC levels between the aphid biotypes, and found little difference in 5mC levels between the biotypes, but much higher levels of 5hmC in the virulent SAM. RWA had two homologs of each of the DNA methyltransferases 1 (DNMT1a and DNMT1b) and DNMT3s (DNMT3a and DNMT3b), but only a single DNMT2, with only the expression of DNMT3 that differed significantly between the two RWA biotypes. RWA has a single ortholog of Ten eleven translocase (DnTET) in the genome. Feeding studies show that the more virulent RWA biotype SAM upregulate DnDNMT3 and DnTET in response to wheat expressing antibiosis and antixenosis.

Mortality, teratogenicity and growth inhibition of three glyphosate formulations using Frog Embryo Teratogenesis Assay-Xenopus.

Ample evidence around the world exists suggesting a link between exposure to glyphosate, toxicity and perturbed physiological functions in non-target organisms. Although glyphosate formulations are widely used for weed and alien plant management, their ecotoxicological information remain scanty. Using the 96-hour Frog Embryo Teratogenesis Assay-Xenopus protocol, embryotoxicity and teratogenicity of three glyphosate-based formulations were assessed. Embryos of Xenopus laevis were exposed to Roundup, Kilo Max and Enviro Glyphosate at concentration of 0.3-1.3, 130-280 and 320-560 mg acid equivalent (a.e.)/L respectively. The results showed Roundup to be more toxic than the other formulations with a 96-hour LC50 of 1.05 mg a.e/L. compared with 207 mg a.e./L, and 466 mg a.e./L for Kilo Max and Enviro Glyphosate respectively. Although, both Roundup and Kilo Max formulations show inhibition on growth of the embryo-larva (P Ë‚ .05), the minimum concentration inhibiting growth ratios of the three formulations was >0.30 baseline, indicating no significant growth inhibiting effect in the formulations. For teratogenicity, Roundup and Enviro Glyphosate formulations exhibited increasing teratogenic traces, with the teratogenic index at 1.7 and 1.6 respectively. Kilo Max formulation shows low teratogenicity with the teratogenic index at 1.4. Characteristic malformation induced by these formulations included generalized edema, cardiac and abdominal edema, improper gut formation and axial malformations. This study confirms that these formulations could be a potential physiological and ecological health disruptor, particularly concerning teratogenicity and growth disruption. Further studies to characterize the contributions of their surfactants will be invaluable.

An evaluation of the endocrine disruptive potential of crude oil water accommodated fractions and crude oil contaminated surface water to freshwater organisms using in vitro and in vivo approaches.

Knowledge regarding the potential impacts of crude oil on endocrine signaling in freshwater aquatic vertebrates is limited. The expression of selected genes as biomarkers for altered endocrine signaling was studied in African clawed frog, Xenopus laevis, tadpoles and juvenile Mozambique tilapia, Oreochromis mossambicus, exposed to weathered bunker and unweathered refinery crude oil water accommodated fractions (WAFs). In addition, the expression of the aforementioned genes was quantified in X. laevis tadpoles exposed to surface water collected from the proximity of an underground oil bunker. The (anti)estrogenicity and (anti)androgenicity of crude oil, crude oil WAFs, and surface water were furthermore evaluated using recombinant yeast. Thyroid hormone receptor beta expression was significantly down-regulated in X. laevis in response to both oil WAF types, whereas a further thyroid linked gene, type 2 deiodinase, was up-regulated in O. mossambicus exposed to a high concentration of bunker oil WAF. In addition, both WAFs altered the expression of the adipogenesis-linked peroxisome proliferator-activated receptor gamma in X. laevis. The crude oil and WAFs exhibited antiestrogenic and antiandrogenic activity in vitro. However, O. mossambicus androgen receptor 2 was the only gene, representing the reproductive system, significantly affected by WAF exposure. Estrogenicity, antiestrogenicity, and antiandrogenicity were detected in surface water samples; however, no significant changes were observed in the expression of any of the genes evaluated in X. laevis exposed to surface water. The responses varied among the 2 model organisms used, as well as among the 2 types of crude oil. Nonetheless, the data provide evidence that crude oil pollution may lead to adverse health effects in freshwater fish and amphibians as a result of altered endocrine signaling. Environ Toxicol Chem 2017;36:1330-1342. © 2016 SETAC.