Circadian clock outputs regulating insect photoperiodism: A potential role for glutamate transporter.

Many temperate ectotherms survive winter by entering diapause - a state of developmental (or reproductive) suppression or arrest - in response to short autumnal day lengths. Day lengths are assessed by the circadian clock, the biological time-keeping system that governs biological rhythms with a period of approximately 24 h. However, clock output molecules controlling this photoperiodic response are largely unknown for many insects. To identify these molecules in Hemiptera, we performed RNAi knockdowns of several candidate genes in the bean bug Riptortus pedestris to determine whether their silencing affects photoperiodic regulation of ovarian development (reproductive diapause). Knockdown of diuretic hormone 31, short neuropeptide F, neuropeptide F, ion transport peptide, neuropeptide-like precursor 1, and choline acetyltransferase had no effect on ovarian development and were therefore ruled out as regulators of the photoperiodic response. However, knockdown of vesicular glutamate transporter promoted ovarian development under diapause-inducing short days, and this is the first report of the functional involvement of glutamate signalling in insect photoperiodism. Improved knockdown of this transporter (or receptor) and RNAi of other genes involved in glutamate signal transduction is required to verify its role as an output of the circadian clock.

Mapping PERIOD-immunoreactive cells with neurons relevant to photoperiodic response in the bean bug Riptortus pedestris.

Circadian clock genes are involved in photoperiodic responses in many insects; however, there is a lack of understanding in the neural pathways that process photoperiodic information involving circadian clock cells. PERIOD-immunohistochemistry was conducted in the bean bug Riptortus pedestris to localise clock cells and their anatomical relationship with other brain neurons necessary for the photoperiodic response. PERIOD-immunoreactive cells were found in the six brain regions. In the optic lobe, two cell groups called lateral neuron lateral (LNl) and lateral neuron medial (LNm), were labelled anterior medial to the medulla and lobula, respectively. In the protocerebrum of the central brain, dorsal neuron (Prd), posterior neuron (Prp), and antennal lobe posterior neuron (pAL) were found. In the deutocerebrum, antennal lobe local neurons (ALln) were detected. Double immunohistochemistry revealed that PERIOD and serotonin were not co-localised. Furthermore, pigment-dispersing factor-immunoreactive neurons and anterior lobula neurons essential for R. pedestris photoperiodic response were not PERIOD immunopositive. LNl cells were located in the vicinity of the pigment-dispersing factor immunoreactive cells at the anterior base of the medulla. LNm cells were located close to the somata of the anterior lobula neurons. Fibres from the anterior lobula neurons and pigment-dispersing factor-immunoreactive neurons had contacts at the anterior base of the medulla. It is suggested that LNl cells work as clock cells involved in the photoperiodic response and the region at the medulla anterior base serves as a hub to receive photic and clock information relevant to the photoperiodic clock in R. pedestris.

Afferent neural pathways from the photoperiodic receptor in the bean bug, Riptortus pedestris.

Adult diapause in the bean bug, Riptortus pedestris, is controlled by the photoperiod, which is received by retinal cells in the central region of the compound eyes. To resolve the afferent neural pathways involved in the photoperiodic response, we examine fibre projections from the photoperiodic receptors to the brain and investigate the roles of the posterior optic tract (POT) in the photoperiodic response. Reduced-silver impregnation and synapsin immunolabelling revealed that the medulla was divided into nine strata: the outer layer comprises 4 strata, the inner layer comprises 4 strata and a serpentine layer separates the inner and outer layers. Biotin injection revealed that retinal fibres from the central region of the compound eye terminated in either the central part of the lamina or the central part of the medulla 3rd or 4th layer. Biotin injection into the central part of the medulla labelled 5 distinct afferent pathways: two terminated in a region of ipsilateral anterior protocerebrum, while the other three had contralateral projections. One pathway ran through the POT and connected to the bilateral medulla serpentine layers. When the POT was surgically severed, diapause incidence under short-day conditions was significantly reduced compared to that observed following a sham operation. However, an incision at a posterior part of the medulla and lobula boundary, as a control experiment, did not affect the photoperiodic response. These results suggest that photoperiodic signals from the central region of the compound eye are transferred to neurons with fibres running in the POT for photoperiodic response in R. pedestris.