Neither per, nor tim1, nor cry2 alone are essential components of the molecular circadian clockwork in the Madeira cockroach

Abstract

Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNA i) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clocḱ s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels withiñ two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rḿ per RNAi or Rm´cry2 RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´per RNAi and shorter periods for Rm´tim1 RNAi and Rm´cry2 RNAi .As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.

Model and Non-model Insects in Chronobiology

“…purely on photoreception via the compound eyes (Komada et al, 2015;Kutaragi et al, 2018). In other species that lack cry1 (i.e., beetles and cockroaches), tim1 may play a different role in the circadian rhythm generation (Werckenthin et al, 2012(Werckenthin et al, , 2020Li C-J et al, 2018), which stresses the complexity in evolution of divers sets of clock genes in different insects. Recently, a model which includes two different pacemakers with different clock gene sets was proposed for the cockroach to explain per, cry2, and tim1 function in parallel (Werckenthin et al, 2020) (see Figure 1).…”

“…In other species that lack cry1 (i.e., beetles and cockroaches), tim1 may play a different role in the circadian rhythm generation (Werckenthin et al, 2012(Werckenthin et al, , 2020Li C-J et al, 2018), which stresses the complexity in evolution of divers sets of clock genes in different insects. Recently, a model which includes two different pacemakers with different clock gene sets was proposed for the cockroach to explain per, cry2, and tim1 function in parallel (Werckenthin et al, 2020) (see Figure 1). The cockroach clock may work with three regulatory loops, because knock-down of neither per, nor tim1 nor cry2 alone was successful to induce arrhythmic behavior in the animals (Werckenthin et al, 2020).…”

“…Recently, a model which includes two different pacemakers with different clock gene sets was proposed for the cockroach to explain per, cry2, and tim1 function in parallel (Werckenthin et al, 2020) (see Figure 1). The cockroach clock may work with three regulatory loops, because knock-down of neither per, nor tim1 nor cry2 alone was successful to induce arrhythmic behavior in the animals (Werckenthin et al, 2020). Interestingly, Hymenoptera are so far the only insect group that lack both, cry1 and tim1 (only cry2 and tim2 are present; see also section "The relevance of Zeitgebers differs between flies and bees"), and they display a clock gene set that is more similar to the mammalian clock (Rubin et al, 2006;Sandrelli et al, 2008).…”