The circadian clock and photoperiodic timer of the linden bug, Pyrrhocoris apterus, were systematically explored by a combination of microsurgical intervention, gene silencing, and we have generated stable genetic mutations for a subset of genes. Using a combination of these approaches, we were able to identify a new insect clock gene. At the same time, we pointed to unexpected role of Drosophila-type timeless (dTIM) in insects. Drosophila-type timeless (often just timeless in the Drosophila community) is a key component of the circadian machinery, the absence of which results in a completely nonfunctional clock. However, the circadian clock of the linden bug, Pyrrhocoris apterus, remains robustly functional even when dTIM is completely removed. The altered “ticking” in the dTIM mutant confirms that dTIM is a clock component, but its function unexpectedly differs. This non-canonical role of dTIM explains possible transitions leading on the one hand to the clock setup known in humans and on the other hand to the clock setup known in Drosophila.
The role of circadian clock genes and outputs of the photoperiodic timer was addressed at several levels:
Firstly, a systematic analysis of the photoperiodic response was determined for genetically engineered mutants in mammalian-type of cryptochrome (m-cry) gene. Although the mutants almost lost their ability to diapause, a subset of females was still able to distinguish between long and short days (photoperiods). This phenotype correlates with partially rhythmic clock of m-cry mutants.
Secondly, we engineered mutants in Pigment Dispersing Factor (PDF) gene in the linden bug P. apterus (the first mutants in PDF outside of Drosophila). PDF is a key neuropeptide in Drosophila circadian clock. Our data confirm that PDF is a part of the circadian machinery in the linden bug. Surprisingly, PDF mutants show an aberrant inversion of the locomotor activity to night. Importantly, the photoperiodic timer was heavily disrupted by depletion of PDF. However, even null mutants still discriminated between photoperiods. These results suggest that the photoperiodic timer is a neuronal network property.
Thirdly, we addressed the output of the photoperiodic timer, the reproductive diapause and specifically addressed the male- and female-specific traits. In females, the absence of juvenile hormone (JH) is a hallmark of reproductive diapause. Analysis of male reproduction revealed a surprising redundancy in the system. While JH is a potent diapause terminator in males acting through its canonical receptors, the photoperiodic termination of diapause in males is JH-independent.
One of the major signaling pathways in insects includes insulin-like peptides. We have shown that insulin signaling regulates wing polyphenism in P. apterus, where long-winged morphs are more frequent in summer and show reduced/delayed reproduction. Furthermore, the presence of three insulin receptors (InR) in P. apterus promoted our systematic analysis of evolution of InR across insects, where we identified two gene clusters (Clusters I and II) resulting from an ancestral duplication in a late ancestor of winged insects, which remained conserved in most lineages. One remarkable yet neglected feature of InR evolution is the loss of the tyrosine kinase catalytic domain, giving rise to decoys of InR in both clusters. Importantly, we described a new tyrosine kinase-less gene (DR2) in the Cluster II, conserved in apical Holometabola for more than 300My.
The geographic variability in circadian clocks, photoperiodic timer and related phenomena were explored in several parallel experiments. Comparison of physiological properties in adult bugs revealed geographic variability in supercooling points, indicating latitudinal cline in cold hardiness in the linden bug. Genetic experiments were applied to identify components responsible for geographic variability in circadian clocks and to identify the genetic basis of diapause regulation. We have also isolated seven non-diapause mutant lines from geographically distant field-lines.
