engleski

Circadian gene expression analysis in non-model insects

Freshwater benthic macrofauna consists of many insects exhibiting interesting chronobiological phenomena, i.e. cyclic physiological and ethological adaptations to solar- and lunar- related rhythms (circadian rhythms). Since cellular processes are largely regulated by circadian rhythms, the adaptation of organisms' circadian rhythm to the environment is of vital importance for perservation of ecosystem biodiversity. Freshwater insects are largely non- model insects, which makes the analyses of their transcriptome very challenging. Developing genetic tools for non-model insect taxa could advance the exciting field of insect photoperiodism. The aim of this study was to determine the best way to preserve nucleic acids of archive macrozoobenthos samples, and to develop a method for gene expression analysis of those organisms. Individuals belonging to 7 different non-model species from archive collections (1992, 2008, 2016 and 2020) were used. Drosophila melanogaster was selected as a control (model-insect) sample. Based on Drosophila genome, specific primers were selected for Clk gene amplification. Non-model organisms were taken from the fixative they were stored in, and embedded in paraffin blocks. After the RNA isolation from tissue sections, reverse transcription was performed and expression of Clk gene was analysed by real time polymerase chain reaction. All samples (species) used in this study showed amplification of Clk gene. Such results show the possibility of evolutionarily preserved genes’ analysis, such as circadian genes, using primers designed based on the sequence of the related model organism. Furthermore, this finding opens the possibility of using archive collections in long-term monitoring of urban streams at the molecular level.

freshwater benthic macrofauna ; circadian rhythm ; gene expression

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