Work Package 4 (ESR 5)

Natural variation and genetic architecture of biological timing

General details

Czech RepublicLocation
Biologicke centrum AV CR, v.v.i.
Czech Republic

Supervisors
D. Dolezel

ESR 5
Lenka Chodáková

Other partners involved
University of Wuerzburg (DE)

Secondments

University of Wuerzburg for 3 months in year 1 – Neuroanatomy of Linden bug
University Groningen for 3 months in year 2 – QTL analysis of diapause in Linden bug
Actual Analytics for 1 month in year 2 – Test software in Linden bug

Objectives

  • • Localize the products of circadian genes using in situ hybridization and ICC in the brain and body of linden bug (with Förster).
  •  Explore the role of several candidate genes by using transient RNAi approach and explore geographic variability of these genes in established collection of field-lines originating from localities of different latitudes and altitudes (with Van de Zande, Hut and Beersma).
  • • Develop (with Actual) an automated behavioural analysis to characterize diapause/non-diapause quantitatively and qualitatively.

Methods

  • Test available antibodies for Linden bug and perform in situ hybridization by month 18
  • Characterize field-lines and prepare markers for QTL analysis by month 26
  • Functional validation of clock genes in diapause and circadian rhythms by RNAi – month 36

Description

Diapause and circadian mechanisms in the linden bug
The linden bug Pyrrhocoris apterus is phylogenetically distant from the most commonly used insect model species such as Diptera, Lepidoptera and Hymenoptera which makes it a valuable addition to studies aimed at unravelling the evolution of circadian clocks. It exhibits a robust reproductive diapause induced by shortening photoperiod. We have recently established a wide collection of field-lines originating from localities of different latitudes and altitudes. These lines exhibit a clear clinal variability in the photoperiodic response curves, diapause-inducing photoperiod and also in their circadian free running periods.  We have also isolated three mutant lines which are insensitive to photoperiodic changes. Our key questions are: 1) Do photoperiodic and circadian mechanisms share the same genes and brain structures? 2) Which genes are responsible for geographic variability in circadian and photoperiodic clocks? Will all or only some photoperiodic mutants also exhibit altered circadian behaviour and vice versa? With co-supervisors Helfrich-Forster and Pyza we will localize the products of circadian genes using in situ hybridization and immunocytochemistry (ICC) in the brain and body. Using microsurgical manipulations we will test which brain structures are essential for circadian and photoperiodic behaviour and determine the anatomical substrate for light input into these two time-keeping devices. Since reduced locomoter activity is associated with diapause inP. apterus, we will develop (with Actual) an automated rhythm behaviour analysis to characterize diapause/non-diapause behaviour quantitatively and qualitatively. The secondment at Oxitec will be important for learning transgenesis techniques as a possible integrative reverse genetic approach in non-model organism. We will explore the role of several candidate genes by using our current transient RNAi approach, which is established in P. apterus. The geographic variability in photoperiodic and circadian phenotypes will be used as a basis for mathematical modelling of these two clocks (with Beersma and Hut) and predictions stemming from these models will be experimentally tested. We will use Representational Difference Analysis (RDA) and SNPs to map 3 existing photoperiodic mutations. QTL analysis (with van de Zande and Beukeboom) as well as candidate gene sequencing will reveal the genetic basis of the geographic variability in photoperiodic and circadian clocks of P. apterus. Interspecific sequence/functional comparisons among P. apterusNasonia, and Drosophila genes will provide evolutionary insights into the circadian-diapause relationship.

Results and milestones

1) Month 8 Project plan & personal development plan for individual training requirements
2) Month 14 First year ESR5 report
3) Month 24 Finish and submit characterization of circadian genes in Pyrrhocoris apterus by month 24
4) Month 36 At least one publication drafted

Synergies, Risks & Exploitation

The partners will apply their various expertises to generate a multidimensional approach to study this non-model insect. RDA is quite a difficult method but we have enough expertise among us to succeed. Results can be used by SMEs to enhance field trials and increase production of sterile males.

See more Work Packages in Research Area A