The role of Daphnia pulex micrornas in response to short-term and multi-generational cadmium acclimation
Cadmium (Cd) is a non-essential heavy metal that is highly toxic to aquatic organisms. Daphnia pulex, a keystone species in freshwater ecosystems, are known for their quick responsiveness and ability to develop tolerance to Cd. Recent research with plants and vertebrates suggests microRNAs (miRNA), a family of short non-coding RNAs, are involved in the response and development of tolerance to heavy metals including Cd. However, no comparable studies have been carried out with invertebrates. Further, at the time this research began, only 44 miRNAs had been predicted in D. pulex and none validated. In order to reveal the role of miRNAs in D. pulex response and tolerance to Cd, I carried out a series of experiments aimed at: 1) predicting and validating D. pulex miRNAs; 2) identifying miRNAs related with short-term and multi-generational Cd acclimation; and 3) predicting gene targets and their potential role in response to Cd exposure. To achieve these goals, I first computationally predicted 75 D. pulex miRNAs, validated 8 in different developmental stages, and identified a stably expressed reference gene during D. pulex development. MiRNA expression profiles suggests miRNAs play critical roles during D. pulex development. Secondly, I used a miRNA microarray to identify Cd-responsive miRNAs under short-term (48 h) Cd exposure. A total of 29 Cd-responsive miRNAs were identified and predicted gene targets suggests these miRNAs are involved in ion transport, mitochondrial damage, DNA repair and cellular energy balance. In addition, Cd-responsive miR-210 was shown to be hypoxia-responsive and I proposed both Cd and hypoxia induce miR-210 via a similar HIF1α related-pathway. Moreover, the microarray detected 230 robustly expressed miRNAs. I used this data to predict miRNA gene targets using thermodynamics and complementary seed region methods and stored this information in a public online database (www.daphniatar.org). Finally, I exposed D. pulex to sub-lethal Cd concentrations for 25 generations to induce Cd tolerance and observed the maintenance of enhanced tolerance in a Cd-free environment for an additional three generations. Cd tolerance was related with 10 miRNAs that target genes that can switch cellular energy allocation by suppressing GTPase and cuticle protein pathways. In addition, this experiment showed enhanced Cd tolerance is related with induction of metallothioneins MT3 and MT4 and revealed D. pulex can switch energy allocation to growth and reproduction in Cd-free environments by down-regulating MT1 and MT3. In conclusion, this research provides novel information on the molecular mechanisms responsible for short-term and multi-generational Cd exposure in aquatic invertebrates.
Nichols, Purdue University.
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