Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus

Abstract

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila, PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus, an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect’s circadian and photoperiodic systems.

Morphology and synaptic connections of pigment‐dispersing factor‐immunoreactive neurons projecting to the lateral protocerebrum in the large black chafer, Holotrichia parallela

“…The lack of LA PDF neurons has also been reported in other insects, including the goldsmith beetle (Závodská et al, 2003), the backswimmer (Závodská et al, 2003), bees (Beer et al, 2018;Weiss et al, 2009;Závodská et al, 2003), bugs (Hasebe et al, 2022;Ikeno et al, 2014;Kotwica-Rolinska et al, 2022;Vafopoulou et al, 2010), the bristletail (Závodská et al, 2003), the caddisfly (Závodská et al, 2003), the damselfly (Závodská et al, 2003), flies (Helfrich-Förster, 1997;Nässel et al, 1993;Závodská et al, 2003), the praying mantis (Sehadová et al, 2003), the stonefly (Závodská et al, 2003), and the waterstrider (Závodská et al, 2003). The presence of LA PDF neurons appears to be species-specific, and it would be reasonably assumed that they have roles specialized to certain species.…”

“…The functional role of PDF in phase shift suggests that it internally coordinates bilaterally located pacemaker cells. The crucial role of PDF in generating the circadian rhythm has also been demonstrated in the German cockroach (Lee et al., 2009) and linden bug (Kotwica‐Rolinska et al., 2022). Finally, in the honeybee, there are PER‐ and PDF‐ir lateral neuron 2 corresponding to the LN v of the fruit fly, in which the intensity of PER immunolabeling exhibits a circadian oscillation in both the forager and nurse (Fuchikawa et al., 2017).…”

“…The l-LN v connects bilateral optic lobes through an axon in the posterior optic tract to extend the wide range of fibers over the entire surface area of the bilateral ME neuropils, whereas the processes of the PDF-ir s-LN v are restricted in the ipsilateral hemisphere, and it projects the axon terminals exclusively in the lateral protocerebrum and bears the dendritic arbors within the AME (Helfrich-Förster, 1997;Helfrich-Förster et al, 2007). A group of ME PDF neurons of the large black chafer extends the ventrally elongated processes near the AME (Figure 3a-c) and their axons connecting the bilateral optic lobes run in the dorsal protocerebrum (Figure 5), a bundle of axons linking both optic lobes with a similar trajectory also seen in the bugs (Hasebe et al, 2022;Ikeno et al, 2014;Kotwica-Rolinska et al, 2022). Those fibers in the large black chafer are reminiscent of the ventral elongation in the AME and posterior optic tract in the fruit fly that exclusively stem from the l-LN v cell (Helfrich-Förster et al, 2007).…”

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila

“…K224 region indicates whether NKRQK or TKRQK motif was found in the region corresponding to the 220–224 position within the catalytic domain of D. melanogaster DBT. Major changes in the circadian clock setup are depicted: presence of CRY mammalian (mCRY) type, loss of TIMELESS-drosophila type (dTIM), loss of PERIOD (PER) ( Kotwica-Rolinska et al, 2022b ), and transition of BMAL (activation domain is present) to CYC (activation domain lost) ( Supplementary Figure S8 ). DBT-interacting protein paralogs BRIDE of DBT (BDBT) was found only in Diptera (for details and BDBT phylogeny see Figure 5 ).…”

“…Therefore, we are reaching technical limitations of ds RNA mediated interference, even though a 288 bp long dsRNA was successfully used to knock down isoform-specific transcripts ( Bajgar et al, 2013 ). An alternative approach could utilize gene editing, a method established and used for circadian research in P. apterus ( Kotwica-Rolinska et al, 2019 ; Kotwica-Rolinska et al, 2022b ).…”