To understand the role of gene expression in adaptive variation, it is necessary to examine expression variation in an ecological context. Quantitative real-time PCR (qPCR) is considered the most accurate and reliable technique to measure gene expression and to validate the data obtained by RNA-seq; however, accurate normalization is crucial. In Chile, the freshwater silverside fish Basilichthys microlepidotus inhabits both polluted and nonpolluted areas, showing differential gene expression related to pollution. In this study, we infer the stability of six potential reference genes (tubulin alpha, hypoxanthine-guanine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, 60S ribosomal protein L13, and 60S ribosomal protein L8) in the gills and liver of silverside individuals inhabiting polluted and nonpolluted areas. To validate the reference genes selected, the most and least stable reference genes were used to normalize two target transcripts, one for each organ. The RefFinder tool was used to analyze and identify the most stably expressed genes. The 60S ribosomal protein L8 gene was ranked as the most stable gene for both organs. Our results show that reference gene selection influences the detection of differences in the expression levels of target genes in different organs and, also highlighting candidate reference genes that could be used in field studies.

How organisms adapt to unfavorable environmental conditions by means of plasticity or selection of favorable genetic variants is a central issue in evolutionary biology. In the Maipo River basin, the fishBasilichthys microlepidotusinhabits polluted and non-polluted areas. Previous studies have suggested that directional selection drives genomic divergence between these areas in 4% of Amplified Fragment Length Polymorphism (AFLP) loci, but the underlying genes and functions remain unknown. We hypothesized thatB. microlepidotusin this basin has plastic and/or genetic responses to these conditions. Using RNA-Seq, we identified differentially expressed genes in individuals from two polluted sites compared with fish inhabiting non-polluted sites. In one polluted site, the main upregulated genes were related to cellular proliferation as well as suppression and progression of tumors, while biological processes and molecular functions involved in apoptotic processes were overrepresented in the upregulated genes of the second polluted site. The ornithine decarboxylase gene (related to tumor promotion and progression), which was overexpressed in both polluted sites, was sequenced, and a parallel pattern of a heterozygote deficiency and increase of the same homozygote genotype in both polluted sites compared with fish inhabiting the non-polluted sites was detected. These results suggest the occurrence of both a plastic response in gene expression and an interplay between phenotypic change and genotypic selection in the face of anthropogenic pollution.

Daphnia exilis is a halophylic species that was recently found in Chile, distant from its natural range. In this study, we analyze the osmoregulatory and life-history responses to salinity exhibited by Daphnia exilis, as a first step towards understanding the ecology of this exotic species whose invasion potential has been related to its ability to tolerate saline environments. A set of laboratory experiments were carried out, in which we exposed organisms to a salinity gradient, and measured internal and environmental osmolality, assessed the effect of acclimation time and measured life-history traits that were used to perform a demographic retrospective analysis. Our results showed that (i) D. exilis exhibited a hyper-osmoconformer response, (ii) acclimation time did not exert effects on the osmoregulatory response, (iii) salinity up to 6 g L⁻¹ did not alter the population growth rate, (iv) at 8 g L⁻¹ population growth rate declined, mainly due to a delayed age at maturity and reduced fertility. Based on this information, we suggest that the responses to salinity exhibited by the exotic D. exilis confer an advantage over its natural enemies and may facilitate colonization through exploiting disturbed places as chemical refuges.