An integrated approach to the analysis of the circadian clock of the blow fly Lucilia cuprina
The Australian sheep blow fly Lucilia cuprina is an economically important dipteran pest whose circadian behavioural rhythms have been the subject of considerable scrutiny. The underlying biochemical nature of these rhythms however, has remained a mystery. The primary objective of this thesis was therefore to investigate the molecular control of circadian rhythms in L. cuprina using an integrative approach. To these ends, a dynamic molecular simulation model for L. cuprina was formulated using existing biochemical data on insect circadian clocks. The validity of this simulation model was subsequently tested at both molecular and behavioural levels.
The basic molecular assumptions of the simulation model were tested by cloning a full length L. cuprina per cDNA and analysing its mRNA and protein expression levels. Isolation of the 4 Kb L. cuprina per cDNA revealed the conservation of three functional domains known to be important for circadian clock function; namely the PAS dimerisation motif (with 92% identity to D. melanogaster at the amino acid level), and the cytoplasmic and nuclear localisation domains (with 85% and 80% identity respectively). A fourth domain, the threonine-glycine (TG) repeat region, was also found to be conserved, but severely truncated in L. cuprina. No length variation was found in the TG repeat of flies collected from several different latitudinal zones, and no correlation was detected between sequences flanking the repeat and latitude of collection of flies. Thus, the contention that the TG repeat region plays a role in temperature compensation of the circadian clock is cast in doubt. Expression analyses (using quantitative RT-PCR) showed per mRNA levels to undergo diel oscillations with a period (24 h) and peak phase (Zt 12) consistent with the Drosophila data. PER-immunoreactive protein oscillations were also demonstrated, with peak immunoreactivity lagging approximately 3 h behind peak mRNA levels.
The behavioural predictions of the model were tested by recording adult locomotor activity under different light regimes. The simulation model successfully predicted free-run, entrainment, the effect of short light pulses, and the effects of constant lighting on behavioural rhythms. Disparities between the simulated and real phase response cnrves for L. cuprina are hypothesised to be indicative of an ealier nuclear entry time of the PER-TIM dimer in L. cuprina compared with D. melanogaster.
The three different approaches of simulation modellingo molecular analysis and behavioural investigation are integrated in the discussion in order to help provide a comprehensive explanation of circadian function in L. cuprina. The benefits of an integrated approach to the analysis of circadian function are discussed, as is the relevance of the present findings to the development of a clock-based control strategy for this economically important pest species.