Work Package 1 (ESR 1)


DIAPAUSE AND THE CLOCK

General details


UKLocation

University of Leicester, UK

Supervisor
Professor C.P. Kyriacou

ESR
Ane Martín Anduaga

Other partners involved
University of Padova (IT)
University of Groningen (NL)

Secondments

University of Padova, for six months during year 2, to integrate the insulin signalling diapause genes into the evolutionary part of the project.

Objectives

To study polymorphisms in Drosophila clock and diapause genes and their inter relationships. Test clock mutations for their effects on diapause

• Test clock mutations for their effects on diapause
• Genetic dissection of tissues responsible for diapause
• Functional/evolutionary analysis of clock/diapause gene polymorphisms and modelling population dynamics

Methods

Novel ecologically relevant measures of diapause allied to genetic analyses and state of-the-art neurogenetic manipulation using GAL4-UAS methodology, immunocytochemistry (ICC), behavioural analysis, statistics, gene expression. Neutrality tests of gene frequencies and mathematical modelling. Tasks and methodology:

• Phenotypic and statistical analyses of clock mutations
• GAL4-UAS technology to dissect relevant tissues involved in the above effects including gene expression analyses (ICC, qPCR)
• Analysis of spatial distributions of clock and diapause gene polymorphisms using methods from evolutionary and population genetics and mathematical modelling

Description

Molecular-genetic analysis of diapause in D. melanogaster
Analysis of polymorphisms that show latitudinal variation has provided an extremely useful approach in identifying genes that are involved in clock phenotypes and diapause, examples being timeless (tim), period (per), and non-clock genes, couch potato (cpo), and the insulin receptor (InR).  The insulin pathway is extremely important for ageing and diapause (which represents an arrest of ageing), and so we shall be examining natural variation in genes that encode components of this important signalling pathway.  We shall extend these observations by investigating whether cold-temperature splice variants of per and tim genes, and coding polymorphisms within Per that are relevant for circadian thermal phenotypes, might also be involved in diapause.  In addition, from D. melanogaster lines we have collected recently in Europe and North America, we shall be able to examine polymorphisms in candidate genes for latitudinal patterns, and test their adaptive significance using a battery of neutrality tests and other population genetic approaches that will investigate the spread of any gene under selection, calculate selection coefficients and migration rates. Any gene that appears to be under selection can then be studied for its functional implications for diapause or other circadian traits that may be under latitude-based environmental selection pressure. Although mutations in clock genes can disrupt diapause in D. melanogaster, whether these genes are directly involved in diapause via the clock mechanism or whether these are indirect pleiotropic effects, has been hotly disputed.  We shall directly test the hypothesis by ablating subgroups of clock neurons and associated tissues, such as external and internal photoreceptors, using various combinations of Gal4 drivers and UAS constructs and examine whether these manipulations disturb diapause.

Results and milestones

• Project & personal development plan for individual training requirements (end of year 1)
• Diapause clock genetics report (end of year 1)
• Neurogenetics of diapause report (end of year 2)
• Population studies report (end of year 3)

Synergies, Risks and Exploitation

University of Leicester and University of Padova’s expertise in the different methodologies plus Groningen’s in mathematical modelling generate a multidimensional attack on this problem. Exploitation: Scientific publication, new collaborative grants (SPNCG). Private sector gains knowledge of how polymorphisms that affect developmental strategies influence insect release programmes

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