Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

Assessment of oxidative stress biomarkers in the threatened annual killifish Austrolebias charrua exposed to Roundup.

This study aimed to analyze the toxic effects of Roundup Transorb® on the endangered Neotropical annual killifish Austrolebias charrua through the assessment of molecular and biochemical biomarkers. The fish were collected in temporary ponds and exposed to environmentally realistic concentrations of the herbicide (5 mg.L-1 for 96 h). The production of ROS, lipid peroxidation, DNA damage, and membrane fluidity were evaluated in the blood cells by flow cytometry. The mRNA expression of the antioxidant-related genes sod2, cat, gstα, atp1a1, gclc, and ucp1 across the brain, liver, and gills was quantified. The acute exposure of annual killifish to Roundup significantly increased ROS production, lipid peroxidation, and DNA damage in their erythrocytes. Likewise, Roundup Transorb® decreased membrane fluidity in the blood cells of the exposed fish. Gene expression analysis revealed that Roundup exposure alters the relative expression of genes associated with oxidative stress and antioxidant defense. Our results give rise to new insights into adaptive mechanisms of A. charrua in response to Roundup. Since Brazilian annual killifishes strongly risk extinction, this study paves the way for developing novel biotechnologies applied to environmental monitoring and aquatic toxicology assessment.

Probiotic expressing heterologous phytase improves the immune system and attenuates inflammatory response in zebrafish fed with a diet rich in soybean meal.

Although aquaculture is among the fastest growing food production sectors in the world, one of the bottlenecks for the continuity of its expansion is the dependence of animal protein on commercial feed formulations. Vegetable proteins are an alternative due to the low cost and high availability. However, this protein source is accompanied by a series of antinutritional and pro-inflammatory compounds, including phytate. Phytases can be added in feed for phytate degradation and increase nutrient availability. However, the use of purified phytases significantly increases the production costs. An interesting alternative is to use probiotics genetically modified as bioreactors for phytase production. In the present study, a strain of Bacillus subtilis secreting a fungal phytase was used to evaluate the effect of a feed with high content of soybean meal on zebrafish (Danio rerio). We analysed the condition factor (K) of fish, and the expression of genes related to the immune system, inflammatory response and oxidative. stress. The results obtained demonstrate that the transgenic probiotic was efficient in improving the fish condition factor, stimulating the immune system, reducing the inflammatory response and oxidative stress. Thus, probiotics acting as phytase bioreactors can be considered an interesting tool for the adaptation of commercial species to feed of lower cost.