miRNA as early markers of stress conditions

Abstract

Water stress in insects cause lethal effects, and one of the first symptoms are alterations on reproduction, in females typically resulting in oocyte resorption. We propose that miRNAs might be early markers of water stress, and our objective is to identify which miRNAs could be involved in this process using the adult stage of Blattella germanica as model, In this cockroach, when water is suppressed on the third day of adult life, ovarian resorption starts on day five and oviposition fails.

To identify miRNAs involved in oocyte resorption, we performed RNA ultrasequencing and obtained two miRNA libraries from ovaries of five-day-old females, one from control specimens, and the other from females that were water-deprived two days earlier. Sequences from both libraries were analysed in order to find differentially expressed miRNAs.

Among the canonical miRNAs that showed the most striking change in expression, we chose miR-34, which has been described as an important homeostatic miRNA, and miR-7, which confers robustness against environmental changes. We also studied two miRNAs with unknown functions: miR-1692 and miR-1937, because they show a high number of copies in the library of stressed cockroaches. Using quantitative real time PCR (qRT-PCR), we analyzed the expression pattern of these miRNAs in the ovary during the first gonadotrophic cycle, in controls as well as in water-stressed specimens. We also silenced them by injecting antisense miRNAs (anti-miRNAs), which lead to a delay in oviposition and ootheca formation.

Using RNAhybrid, we predicted possible target mRNAs for these miRNAs. By qRT-PCR we also studied the expression levels of these mRNAs in the ovary during the cycle, in controls and in water-stress specimens, and in specimens with silenced miRNAs. Taken together, the whole data suggest that miRNAs are involved in the observed ovarian resorption caused by water stress.