Periodic Reporting for period 2 - InPhoTime (Insect Photoperiodic Timer)
Reporting period: 2018-10-01 to 2020-03-31
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.