C-2006: Evaluating MicroRNA Expression in Two Cold-hardy Gall-forming Insects

Abstract

Eurosta solidaginis and E. scudderiana have been studied as insect models of cold tolerance. However, both species employ different strategies to survive deep, sub-zero temperatures. E. solidaginis is freeze tolerant, allowing the freezing of up to 60% of body water content, whereas E. scudderiana is freeze-avoiding, and prevents the formation of ice. While both enter states of reduced metabolic activity, the mechanisms by which these changes occur are not yet well understood. MicroRNAs have been shown to be capable of regulating protein expression on a vast scale, and have been involved in numerous cellular processes. This makes them ideal candidates as potential regulators of cold hardiness in gall forming insects. Our group has previously shown miR-1 to be over expressed in cold stressed E. solidaginis (Lyons et al., 2013). To investigate the role of microRNAs, larvae from both E. solidaginis and E. scudderiana were incubated at 5 °C for two weeks. A sub-group of larvae were gradually chilled and kept at −15 °C for two weeks. RNA extraction was accomplished via mirVana© RNA extraction kit. Both High-throughput Next Generation Sequencing and qRT-PCR were used to evaluate miRNA expression levels. Of interest, miR-92b was found to be 1,5-fold up-regulated ( P = 0,02) in cold-stressed vs. control larvae of E. solidaginis . A subsequent in silico approach using the PicTarFly microRNA target prediction software (Grun et al., 2005) has shown that miR-92b has the potential to target Carnitine Palmitoyl Transferase, which has been shown to have reduced activity in overwintering E. solidaginis larvae (Joanisse and Storey, 1996). These data represent the first steps in evaluating microRNA involvement in the cold response of gall-forming cold-hardy insects.