N-acetyltransferase (nat) Is a Critical Conjunct of Photoperiodism between the Circadian System and Endocrine Axis in Antheraea pernyi

Abstract

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

Identification of the prothoracicotropic hormone (Ptth) coding gene and localization of its site of expression in the pea aphid Acyrthosiphon pisum

“…However, no evident differences in staining intensity were observed, although this result should be interpreted cautiously as this methodology is not rigorously quantitative. This contrasts with the rhythmic expression and release of Ptth reported in other insects (Sauman & Reppert, ; McBrayer et al ., ; Vafopoulou et al ., ; Mohamed et al ., ). However, a role of PTTH in the output pathway of the aphid circadian clock cannot be discarded and should be dealt with in future investigations through detailed quantification of expression of the Ptth gene along the day‐night cycle using real time quantitative PCR (RT‐qPCR) assays.…”

“…However, a putative crosstalk between aphid circadian clock neurones and the PTTH neurones remains to be investigated. In this respect, it is worth mentioning that the synthesis and release of PTTH in A. pernyi were shown to be under the control of two pairs of circadian clock neurones that produce a rhythmic expression of insect‐type AANAT and, consequently, a circadian rhythm of melatonin (Mohamed et al ., ). It was also suggested that melatonin activates specific receptors in adjacent neurones that synthesize PTTH (Mohamed et al ., ).…”

“…In this respect, it is worth mentioning that the synthesis and release of PTTH in A. pernyi were shown to be under the control of two pairs of circadian clock neurones that produce a rhythmic expression of insect‐type AANAT and, consequently, a circadian rhythm of melatonin (Mohamed et al ., ). It was also suggested that melatonin activates specific receptors in adjacent neurones that synthesize PTTH (Mohamed et al ., ). In a similar way, in vitro experiments in Periplaneta americana showed that melatonin regulated the production and release of PTTH, which, in turn, induced a dose‐dependent release of ecdysteroids by the prothoracic glands that was inhibited by the melatonin receptor antagonist luzindole (Richter et al ., ).…”

“…Diapause is very often a response triggered by day length (photoperiod) variation (Saunders, 2002;Nelson et al, 2009;Ko st al, 2011;Hand et al, 2016). Although the cues that stimulate NSCs to secrete PTTH are known in some insect species (Richter et al, 2000;Mohamed et al, 2014;Wang et al, 2013Wang et al, , 2015, it is not known what factors at the molecular level control the decision to activate or to stop PTTH synthesis and/or release (Vafopoulou & Steel, 1996;Steel & Vafopoulou, 2006;Vafopoulou et al, 2007). Similar to what is known in vertebrates (Dardente et al, 2010), a role for the circadian clock in insect photoperiodic responses has been proposed by several groups (Ikeno et al, 2010;Pegoraro et al, 2014;Denlinger & Armbruster, 2016;Omura et al, 2016;Reppert et al, 2016).…”

Roles of the circadian clock and endocrine regulator in the photoperiodic response of the brown‐winged green bug Plautia stali

“…In these cells, ‘clock genes’ such as period ( per ), timeless ( tim ), cycle ( cyc ) and Clock ( Clk ) are expressed and establish a few interlocked negative‐feedback loops (Tomioka & Matsumoto, ; Tataroglu & Emery, ). Several studies adopt gene silencing using RNA interference (RNAi) directed against clock genes, revealing their involvement in the photoperiodic responses (Sakamoto et al ., ; Ikeno et al ., , , , , ; Mohamed et al ., ; Meuti et al ., ; Mukai & Goto, ; Urbanová et al ., ; Kotwica‐Rolinska et al ., ). For example, Ikeno et al .…”