Journal of Biological Rhythms http://jbr.sagepub.com Photoperiodic Induction of Diapause Requires Regulated Transcription of timeless in the Larval Brain of Chymomyza costata J. Stehlík, R. Závodská, K. Shimada, I. Sauman and V. Kostál J Biol Rhythms 2008; 23; 129 DOI: 10.1177/0748730407313364 The online version of this article can be found at: http://jbr.sagepub.com/cgi/content/abstract/23/2/129 Published by: http://www.sagepublications.com On behalf of: Society for Research on Biological Rhythms Additional services and information for Journal of Biological Rhythms can be found at: Email Alerts: http://jbr.sagepub.com/cgi/alerts Subscriptions: http://jbr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations (this article cites 47 articles hosted on the SAGE Journals Online and HighWire Press platforms): http://jbr.sagepub.com/cgi/content/refs/23/2/129 Downloaded from http://jbr.sagepub.com at OHIO STATE UNIVERSITY LIBRARY on March 31, 2008 © 2008 SAGE Publications. All rights reserved. Not for commercial use or unauthorized distribution. Photoperiodic Induction of Diapause Requires Regulated Transcription of timeless in the Larval Brain of Chymomyza costata J. Stehlík,*,† R. Závodská,*,‡ K. Shimada,§ I. Šauman,*,† and V. Koštál*,†,1 *Institute of Entomology, Biology Centre AS CR, Ceske Budejovice, Czech Republic; †Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic; ‡Pedagogical Faculty, University of South Bohemia, Ceske Budejovice, Czech Republic; §Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan Abstract Photoperiodic signal stimulates induction of larval diapause in Chymomyza costata. Larvae of NPD strain (npd-mutants) do not respond to pho- toperiod. Our previous results indicated that the locus npd could code for the timeless gene and its product might represent a molecular link between circa- dian and photoperiodic clock systems. Here we present results of tim mRNA (real time-PCR) and TIM protein (immunohistochemistry) analyses in the lar- val 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 oscilla- tions 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 responsi- ble 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. Key words: photoperiodism, diapause, seasonal clock, circadian oscillator, timeless gene expression Photoperiodic sensitivity is of primary importance 2007) and transduced into an appropriate adjustment for life-cycle patterning in insects. Seasonal change of of developmental mode: change of the rate of develop- the relative proportion of day-length/night-length is ment and morphology, migration, entrance into dia- perceived by specialized receptors (Shiga and Numata, pause, or various combinations of them all (Tauber 1. To whom all correspondence should be addressed: Vladimír Koštál, Biology Centre of the Academy of Sciences of the Czech ˇ Republic, Institute of Entomology, Branišovská 31, 370 05 Ceské Budeˇjovice, Czech Republic; e-mail: [email protected]. JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 23 No. 2, April 2008 129-139 DOI: 10.1177/0748730407313364 © 2008 Sage Publications 129 Downloaded from http://jbr.sagepub.com at OHIO STATE UNIVERSITY LIBRARY on March 31, 2008 © 2008 SAGE Publications. All rights reserved. Not for commercial use or unauthorized distribution. 130 JOURNAL OF BIOLOGICAL RHYTHMS / April 2008 et al., 1986; Danks, 1987). Diapause is a centrally medi- NPD strain were caused by mutation in a single auto- ated type of dormancy that either represents an oblig- somal gene locus npd (Riihimaa and Kimura, 1989; atory part of ontogeny or is facultatively induced when Riihimaa, 1996). Formal analysis of the photoperiodic specific token stimuli (most often photoperiod) signal clock’s function revealed that an element with a the onset of less favorable season. Diapause allows circadian-oscillatory nature participates in photope- the insects to pass through adverse periods, exploit riodic time measurement (Yoshida and Kimura, 1995; seasonally fluctuating resources, diversify in tropical Koštál et al., 2000b). Daily and circadian oscillations regions, and colonize temperate and polar regions of per mRNA abundance, which were detected in the (Andrewartha, 1952; Danilevskii, 1961; Saunders, 2002; wild-type strain, were missing in the npd mutants Koštál, 2006). (Koštál and Shimada, 2001). The tim mRNA tran- Physiological mechanisms of photoperiodic timing scripts were not detectable by Northern blot analysis of diapause remain little understood (Saunders, 2002; in the fly heads of npd-mutants, whereas they were Saunders et al., 2004; Danks, 2005; Bradshaw and detectable and showed typical daily oscillations in Holzapfel, 2007). A great deal, however, is known the wild-type strain (Pavelka et al., 2003). All these about the molecular basis of the central circadian clock pieces of evidence pointed toward an impaired func- in insects, especially in Drosophila melanogaster (Hall, tion of the central circadian clocks in npd-mutants 2003). Several studies tested the hypothesis on and indicated that the locus npd could code for the whether the known molecular elements of insect cir- tim gene in C. costata. Product of tim gene may thus cadian clocks may serve as functional parts of the pho- represent a molecular link between circadian and toperiodic “calendar” system. Saunders et al. (1989) photoperiodic clock systems in this fly. found that a double deletion of the period (per) failed to Previous studies used C. costata adults. Diapause and prevent the ability of female D. melanogaster flies to photoperiodic sensitivity, however, are expressed only discriminate between long and short days. Despite in larval stages. Hence, the main objective of this that negative finding, investigation into the potential study was to find if there are any differences in the involvement of circadian clock genes in photoperiodic levels and daily/circadian patterns of tim gene response resumed during the last decade. It has been expression and TIMELESS (TIM) protein presence in shown that phases, amplitudes, and/or levels of vari- the brains of the wild-type and npd-mutant larvae. ous clock gene expression were affected by photope- We also present here the analysis of the genomic riod in fleshflies Sarcophaga crassipalpis (Goto and structure of tim gene in both strains. It revealed that Denlinger, 2002) and S. bullata (Goto et al., 2006), lin- npd-mutants carry, in addition to 37 amino acid sub- den bug Pyrrhocoris apterus (Syrová et al., 2003), stitutions, a 1855 bp-long deletion in the 5′-UTR pitcher-plant mosquito Wyeomyia smithii (Mathias region. This deletion removed the start of transcrip- et al., 2005), and silkmoth Bombyx mori (Iwai et al., tion and regulatory motifs E-box and TER-box in the 2006). Recent studies in D. melanogaster and pitcher- promoter. We hypothesize that the deletion was pri- plant mosquito indicated that although circadian marily responsible for suppression of tim transcrip- clocks and photoperiodic calendar appear as geneti- tion in the brain, malfunctioning of the central cally distinct mechanisms/processes, the clock gene circadian clock, and loss of photoperiodic calendar timeless (tim) may affect the incidence of diapause function in npd-mutant larvae of the C. costata. directly, independently of its function in the central circadian oscillator (Mathias et al., 2007; Sandrelli et al., 2007; Tauber et al., 2007). MATERIALS AND METHODS We have contributed to this effort by studying the roles of the clock genes per and tim in photoperi- Insects odism of a drosophilid fly, Chymomyza costata. The mature larvae of this fly enter diapause in response Two strains of Chymomyza costata (Diptera: to subcritically short days (Riihimaa and Kimura, Drosophilidae) were used: a wild-type strain origi- 1989; Koštál et al., 2000a). Riihimaa and Kimura nally collected in Sapporo (43°N), Japan, in 1983 and (1988) selected a mutant strain of flies that did not a NPD mutant strain, which was isolated by respond to a photoperiodic signal and named the Riihimaa and Kimura (1988) from wild-type flies col- strain NPD (Non-Photoperiodic-Diapause). Genetic lected in Tomakomai (42.3°N; ca. 50 km south of linkage analysis confirmed that the larval nonpho- Sapporo, Japan). Larvae, pupae, and adults were cul- toperiodism and adult eclosion arrhythmicity in the tured on an artificial diet of Lakovaara (1969) under Downloaded from http://jbr.sagepub.com at OHIO STATE UNIVERSITY