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.

• Klíčová slova
• CRISPR/Cas9, Pyrrhocoris apterus, circadian clock, cryptochrome-m, diapause, photoperiodic clock, pigment dispersing factor,
• Publikační typ
• časopisecké články MeSH

Cold tolerance is often one of the key components of insect fitness, but the association between climatic conditions and supercooling capacity is poorly understood. We tested 16 lines originating from geographically different populations of the linden bug Pyrrhocoris apterus for their cold tolerance, determined as the supercooling point (SCP). The supercooling point was generally well explained by the climatic conditions of the population's origin, as the best predictor-winter minimum temperature-explained 85% of the average SCP variation between populations. The supercooling capacity of P. apterus is strongly correlated with climatic conditions, which support the usage of SCP as an appropriate metric of cold tolerance in this species.

• Klíčová slova
• cold tolerance, diapause, overwintering, supercooling point,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The Pyrrhocoris apterus (Insecta: Heteroptera) adults attain high levels of cold tolerance during their overwintering diapause. Non-diapause reproducing adults, however, lack the capacity to express a whole array of cold-tolerance adaptations and show relatively low survival when exposed to sub-zero temperatures. We assessed the competence of non-diapause males of P. apterus for responding to heat- and cold-stresses by up-regulation of 70 kDa heat shock proteins (Hsps) and the role of Hsps during repair of heat- and cold-induced injury. PRINCIPAL FINDINGS: The fragments of P. apterus homologues of Hsp70 inducible (PaHsp70) and cognate forms (PaHsc70) were cloned and sequenced. The abundance of mRNA transcripts for the inducible form (qPCR) and corresponding protein (Western blotting) were significantly up-regulated in response to high and low temperature stimuli. In the cognate form, mRNA was slightly up-regulated in response to both stressors but very low or no up-regulation of protein was apparent after heat- or cold-stress, respectively. Injection of 695 bp-long Pahsp70 dsRNA (RNAi) caused drastic suppression of the heat- and cold-stress-induced Pahsp70 mRNA response and the up-regulation of corresponding protein was practically eliminated. Our RNAi predictably prevented recovery from heat shock and, in addition, negatively influenced repair of chilling injuries caused by cold stress. Cold tolerance increased when the insects were first exposed to a mild heat shock, in order to trigger the up-regulation of PaHsp70, and subsequently exposed to cold stress. CONCLUSION: Our results suggest that accumulation of PaHsp70 belongs to a complex cold tolerance adaptation in the insect Pyrrhocoris apterus.

• MeSH
• aklimatizace genetika   MeSH
• hmyz fyziologie   MeSH
• hmyzí proteiny fyziologie   MeSH
• hojení ran genetika   MeSH
• messenger RNA analýza   MeSH
• proteiny tepelného šoku HSP70 genetika   fyziologie   MeSH
• reakce na tepelný šok MeSH
• studené klima MeSH
• teplota MeSH
• upregulace genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• messenger RNA MeSH
• proteiny tepelného šoku HSP70 MeSH