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Insect Photoperiodic Timer

Periodic Reporting for period 2 - InPhoTime (Insect Photoperiodic Timer)

Reporting period: 2018-10-01 to 2020-03-31

The circadian clocks and the photoperiodic timer enable animals to anticipate the daily and the seasonal environmental changes. This ability has contributed to the great success of insects living in temperate regions. Understanding the basic principles and mechanisms regulating insect seasonality belongs to the fundamental biological questions. Moreover, many insect species are important pests or vectors of serious diseases, therefore, understanding their seasonality might have relevant practical consequences for the society. Despite the importance of insect seasonality, the basis of photoperiodic sensing remains elusive, because of the lack of suitable genetic models expressing photoperiod-dependent seasonal phenotypes. Therefore, we have developed the linden bug, Pyrrhocoris apterus, into a genetically tractable model with a robust, photoperiod-dependent reproductive arrest (diapause). This project has three objectives synergistically addressing the architecture of the photoperiodic timer: genetic components, anatomy, and geographic variability.
WP1. The circadian clock of the linden bug, Pyrrhocoris apterus, was systematically explored during the first 30 months of the project. RNA interference was successfully applied to manipulate with the circadian clock machinery. Specific gene-knockdowns resulting in arrhythmic, fast, or slow free running periods were identified. These functional experiments confirmed which canonical circadian clock genes are conserved across taxa, and addressed function of circadian clock genes that are variably present in different insect orders. RNA interference screen was initiated and two novel circadian clock genes were identified. Since circadian clocks are conserved from insects to mammals, at the level of genes and proteins, we expect that the novel circadian clock genes identified in P. apterus are also important components of the clock in other organisms. To further explore circadian clock toolkit, various mutations were introduced into several key clock genes by CRISPR/CAS9 gene editing. In addition to full null mutants (period, timeless, and cryptochrome genes), we have also succeeded in more delicate modification including C-terminal manipulations in CRY2 protein and in epitope tagging of PER protein.
WP2. To decipher neuroanatomy of the photoperiodic timer, microsurgical operations of compound eyes, and neuronal connection between the compound eyes and the brain were identified. Projection from the central medulla highlighted several anatomical pathways that are candidates for the signal input to the photoperiodic timer. Further microsurgical experiments will experimentally test role of the identified anatomical pathways. To further connect anatomy with genetics and physiology, neuropeptides expressed in relevant locations will be identified. Since neuropeptide-encoding genes were mostly unknown in P. apterus, a systematic inventory of neuropeptide-toolkit was identified by prospecting the transcriptome, genome and peptidome. Majority of neuropeptide genes and corresponding receptors known from other insect species were found. Clones for RNA interference were prepared for all neuropeptide transcripts and corresponding receptors. In case of insulin signaling, three insulin receptors were identified in P. apterus. Thorough phylogenetic analysis revealed important general patterns of insulin receptor evolution in insects and functional analysis pointed to role of insulin signaling in wing polyphenism that is characteristic for summer morphs of P. apterus. In addition to ’canonical’ insect peptide neurohormones, systematic search for neuropeptide motifs revealed presence of putatively new insect neuropeptide in P. apterus, the homolog of mammalian TRH. Thus, the complete null mutants were created, antibodies prepared and ligands localized in the central nervous system and the study is prepared for publication. The neuroanatomy of the predicted circuit is further explored using genetic tools, when we created complete null mutant in pigment dispersing factor, a key circadian neuropeptide expressed in neurons connecting both brain hemispheres and compound eyes.
WP3. 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 genetic basis of diapause regulation. Phenotypically characterized offspring from genetic crosses is sequenced and being prepared for DNA analysis in pool-sequencing experiments to identify fixed alleles that are likely responsible for the phenotype. We have also isolated seven non-diapause mutant lines from geographically distant field-lines. The mutant lines are also sequenced and used in genetic experiments to shed light on their origin, molecular basis and possible adaptive value.
The project aspires to reveal genetic basis of insect photoperiodic timers. The progress requires implementation of cutting edge technologies to create unique genetic variants, to identify and manipulate with neuropeptide expression, and to utilize geographic variability in circadian clocks and photoperiodic timers. First goals were successfully achieved during the first 30 months of the project, when inventory of circadian clock genes and neuropeptide repertoire were identified, gene editing was established, and already proven RNA interference was used to identify functional circadian clock genes. Next steps will build on these findings in several complementary experiments. Gene editing will be further optimized and unique genetic variants created and phenotypically characterized. We will specifically create lines where circadian clock runs at faster and slower pace to test any possible connection between the circadian clock and the photoperiodic timer. The systematic RNA interference screen will identify what neuropeptides are connected with reproduction, reproductive diapause, seasonality and circadian clocks. The genetic comparison of non-diapause strains, wild type strains and populations originating from various geographic locations will shed light on the basis of the circadian clocks and the photoperiodic timers. Altogether, this project will reveal fundamental basis of insect seasonality in insects.