Photoperiodic Induction of Diapause Requires Regulated Transcription oftimelessin the Larval Brain ofChymomyza costata

Abstract

Photoperiodic signal stimulates induction of larval diapause in Chymomyza costata. Larvae of NPD strain (npd-mutants) do not respond to photoperiod. Our previous results indicated that the locus npd could code for the timeless gene and its product might represent a molecular link between circadian and photoperiodic clock systems. Here we present results of tim mRNA (real time-PCR) and TIM protein (immunohistochemistry) analyses in the larval brain. TIM protein was localized in 2 neurons of each brain hemisphere of the 4-d-old 3rd instar wild-type larvae. In a marked contrast, no TIM neurons were detected in the brain of 4-day-old 3rd instar npd -mutant larvae and the level of tim transcripts was approximately 10-fold lower in the NPD than in the wild-type strain. Daily changes in tim expression and TIM presence appeared to be under photoperiodic control in the wild-type larvae. Clear daily oscillations of tim transcription were observed during the development of 3rd instars under the short-day conditions. Daily oscillations were less apparent under the long-day conditions, where a gradual increase of tim transcript abundance appeared as a prevailing trend. Analysis of the genomic structure of tim gene revealed that npd-mutants carry a 1855 bp-long deletion in the 5'-UTR region. This deletion removed the start of transcription and promoter regulatory motifs E-box and TER-box. The authors hypothesize that this mutation was responsible for dramatic reduction of tim transcription rates, disruption of circadian clock function, and disruption of photoperiodic calendar function in npd-mutant larvae of C. costata.

Effect of photoperiod on clock gene expression and subcellular distribution of PERIOD in the circadian clock neurons of the blow fly Protophormia terraenovae

“…In C. costata, clear daily oscillations in tim transcription were observed in the third instar larvae under short-day conditions. Daily tim oscillations were less apparent under long-day conditions, whereas a gradual increase in tim transcript abundance appeared to be the prevailing trend (Stehlík et al 2008). In S. crassipalpis, tim mRNA expression was dampened under long-day conditions (Goto and Denlinger 2002).…”

“…However, the critical day length of per 0 is obviously shifted as compared with the wild-type (Saunders et al 1989). Another drosophilid fly, Chymomyza costata, a non-photoperiodic-diapause (npd) strain that does not respond to photoperiod, shows lower levels of tim transcripts and TIM abundance in the larval brain than the wild-type (Riihimaa and Kimura 1988;Stehlík et al 2008). In the flesh fly, Sarcophaga bullata, nondiapause phenotype in pupae and arrhythmicity in eclosion are correlated in a variant strain, and the variant strain has higher levels of per and tim expression than the wild-type (Goto et al 2006).…”

Roles of PER immunoreactive neurons in circadian rhythms and photoperiodism in the blow fly, Protophormia terraenovae

“…In a non-photoperiodic-diapause (NPD) strain of C. costata, of which a single autosomal gene locus encoding tim was mutated, both circadian eclosion rhythms and photoperiodic control of larval diapause were lost (Pavelka et al, 2003). Analysis of tim mRNA and TIM protein in the larval brain indicated that regulated transcription of tim in two brain neurons was required for photoperiodic induction of diapause in C. costata (Stehlik et al, 2008). Studies on D. melanogaster have suggested that tim directly affects the incidence of diapause through circadian photoreception Sandrelli et al, 2007).…”

Developmental trajectories of gene expression reveal candidates for diapause termination: a key life-history transition in the apple maggot flyRhagoletis pomonella

“…Genetic variation in timeless associates with propensity to enter photoperiodically induced diapause in the drosophilids D. melanogaster (Tauber et al, 2007) and Chymomyza costata (Stehlik et al, 2008), and variation in period similarly associates with propensity to diapause in the flesh fly Sarcophaga bullata (Han and Denlinger, 2009). Several gene expression and knockout studies also implicate circadian rhythm genes as potential diapause regulators, although whether the entire pathway is involved remains unresolved (Schiesari et al, 2010).…”