A Computational Analysis Based on Automatic Digitization of Movement Tracks Reveals the Altered Diurnal Behavior of the Western Flower Thrips, Frankliniella occidentalis, Suppressed in PKG Expression

The western flower thrips, Frankliniella occidentalis, a worldwide insect pest with its polyphagous feeding behavior and capacity to transmit viruses, follows a diurnal rhythmicity driven by expression of the circadian clock genes. However, it remained unclear how the clock signal triggers the thrips behaviors. This study posed a hypothesis that the clock signal modulates cGMP-dependent protein kinase (PKG) activity to mediate the diurnal behaviors. A PKG gene is encoded in F. occidentalis and exhibits high sequence homologies with those of honeybee and fruit fly. Interestingly, its expression followed a diel pattern with high expression during photophase in larvae and adults of F. occidentalis. It is noteworthy that PKG expression was clearly observed in the midgut during photophase but not in scotophase from our fluorescence in situ hybridization analysis. A prediction of protein–protein interaction suggested its functional association with clock genes. To test this functional link, RNA interference (RNAi) of the PKG gene expression was performed by feeding a gene-specific double-stranded RNA, which led to significant alteration of the two clock genes (Clock and Period) in their expression levels. The RNAi treatment caused adverse effects on early-life development and adult fecundity. To further analyze the role of PKG in affecting diurnal behavior, the adult females were continuously observed for a 24 h period with an automatic digitization device to obtain movement parameters and durations (%) in different micro-areas in the observation arena. Diel difference was observed with speed in RNAi-control females at 0.16 mm/s and 0.08 mm/s, in photo- and scotophase, respectively, whereas diel difference was not observed for the PKG-specific RNAi-treated females, which showed 0.07 mm/s and 0.06 mm/s, respectively. The diel difference was also observed in durations (%) in the control females, more strongly in the intermediate area in the observation arena. Speed and durations in the different micro-areas in mid-scotophase were significantly different from most photophase in the control females, while speed was significantly different mainly during late photophase when comparing effects of control and RNAi treatments in each light phase. Three sequential stages consisting of high activity followed by feeding and visiting of micro-areas were observed for the control females. For RNAi-treated females, the three phases were disturbed with irregular speed and visits to micro-areas. These results suggest that PKG is associated with implementing the diurnal behavior of F. occidentalis by interacting with expressions of the circadian clock genes.