Flumequine causes developmental and reproductive impairments in Daphnia magna: an in vivo whole-transcriptomic approach.

Among the environmental pollutants of emerging interest, veterinary medical products are in the spotlight. At present, the effects that the excessive release of drugs, such as antibiotics, can cause on aquatic ecosystems are still unknown. Among antibiotics, fluoroquinolones (FQ), and in particular flumequine (FLU), have been heavily used in aquaculture and fish farming to cope with bacterial infections [1]. Consequently, FLU can be frequently found in river and marine sediments [2]. FQs are detected in the environment at concentrations ranging from ng/Lup to µg/L [3], with peaks of around mg/L identified in watercourses near fish farms [4]. In a previous study conducted in our laboratory, 2.0 mg/L FLU has been shown to induce phenotypical and reproductive impairments in Daphnia magna [5]. Here we present a second study that aims to understand the molecular mechanisms underneath FLU toxicity. To reach this goal, we assessed chronic toxicity in D. magna exposed to 0.2 mg/L and 2.0 mg/L, and carried out a whole-transcriptome analysis by means of RNA-sequencing (RNA-seq). The chronic test was performed in general accordance to the OECD 211 guideline [6], the endpoints being: reproductivity, mortality and growth rate. Total RNA was then isolated from pool of three daphnids. A total of nine RNA-seq libraries, three per experimental group (i.e., FLU 0.2 mg/L, FLU 2.0 mg/L, CTRL), were constructed and sequenced. Looking at the phenotypical end-points, we observed that the population exposed to 2.0 mg/L showed a significant drop in reproduction (-46.50% offspring per mother per day, p < 0.05), survival (42.55% mortality, p < 0.01), and growth (-8.83% individual growth per day, p < 0.05). On the other hand, the group of daphnids exposed to 0.2 mg/L showed no statistical difference in phenotypical characters. These findings were supported by the RNA-seq results. Indeed, daphnid exposure to 0.2 mg/L FLU resulted in less transcriptomic changes compared with daphnids exposed to 2.0 mg/L FLU (43 vs 357 differentially expressed genes, DEGs; fold change greater than 2 and FDR < 5% as default parameters). Interestingly, most of DEGs identified at the lowest FLU concentration were likewise modulated at the highest concentration but with increasing fold changes, thus suggesting a linear transcriptional dose-response. Subsequently, the enrichment analysis revealed that the majority of DEGs reported in the 2.0 mg/L FLU group are associated with development, growth, egg components, detoxification mechanisms and response to oxidative stress pathways. Interestingly, even though the lowest FLU concentration did not induce relevant phenotypical changes, it significantly affected the expression of a certain number of genes (i.e., 43); moreover, many of them are involved in reproduction, as demonstrated by the enriched pathways of egg constituents. In perspective, for a deeper understanding of molecular mechanisms of FLU toxicity, it is crucial to investigate whether environmentally relevant concentrations of FLU are likely to induce transgenerational effects, possibly involving epigenetic variations (e.g. DNA methylation), too.