RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

• Published in: Journal of insect physiology 2010-12, Vol.56 (12), p.1738-1745
• Main Authors: Danbara, Yoshiki, Sakamoto, Tomoaki, Uryu, Outa, Tomioka, Kenji
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; biochemical mechanisms; Circadian clock; Circadian Clocks - genetics; Circadian Clocks - physiology; circadian rhythm; Clock gene; clock genes; CLOCK protein; complementary DNA; Cricket; Discosoma; diurnal activity; Drosophila; genes; Gryllidae - genetics; Gryllidae - physiology; Gryllus bimaculatus; insect genetics; Insect Proteins - genetics; Insect Proteins - physiology; locomotion; Locomotion - physiology; Male; molecular genetics; Molecular Sequence Data; neurophysiology; nocturnal activity; nucleotide sequences; Oscillatory mechanism; period; Phylogeny; physiological response; Reverse Transcriptase Polymerase Chain Reaction; RNA - chemistry; RNA - genetics; RNA interference; RNA Interference - physiology; RNAi; Sequence Alignment; sequence analysis; species differences; temporal variation; timeless; timeless gene
• ISSN: 0022-1910; 1879-1611
• DOI: 10.1016/j.jinsphys.2010.07.002

[Display omitted] ▶timeless gene is circadianly expressed in the cricket. ▶ dstim knocks-down tim mRNA and disrupts its circadian expression. ▶tim RNAi does not disrupt circadian locomotor rhythms. ▶tim RNAi only shortens free-running period. Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.