Numerous insect species living in temperate regions survive adverse conditions, such as winter, in a state of developmental arrest. The most reliable cue for anticipating seasonal changes is the day-to-night ratio, the photoperiod. The molecular mechanism of the photoperiodic timer in insects is mostly unclear. Multiple pieces of evidence suggest the involvement of circadian clock genes, however, their role might be independent of their well-established role in the daily oscillation of the circadian clock. Furthermore, reproductive diapause is preferentially studied in females, whereas males are usually used for circadian clock research. Given the idiosyncrasies of male and female physiology, we decided to test male reproductive diapause in a strongly photoperiodic species, the linden bug Pyrrhocoris apterus. The data indicate that reproduction is not under circadian control, whereas the photoperiod strongly determines males' mating capacity. Clock mutants in pigment dispersing factor and cryptochrome-m genes are reproductive even in short photoperiod. Thus, we provide additional evidence of the participation of circadian clock genes in the photoperiodic time measurement in insects.

• Klíčová slova
• Circadian clock, Cryptochrome, Photoperiodism, Pigment dispersing factor, Reproductive diapause,
• MeSH
• cirkadiánní hodiny * genetika   fyziologie   MeSH
• cirkadiánní rytmus fyziologie   genetika   MeSH
• diapauza hmyzu genetika   fyziologie   MeSH
• fotoperioda * MeSH
• Heteroptera * genetika   fyziologie   MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• kryptochromy * genetika   metabolismus   MeSH
• mutace * MeSH
• rozmnožování fyziologie   genetika   MeSH
• sexuální chování zvířat fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hmyzí proteiny MeSH
• kryptochromy * MeSH

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

• Klíčová slova
• ATP, COX, NAD+, NADP+, diapause, energy metabolism, gene expression,
• Publikační typ
• časopisecké články MeSH

Circadian clocks are synchronized with the external environment by light and temperature. The effect of these cues on behavior is well-characterized in Drosophila, however, little is known about synchronization in non-model insect species. Therefore, we explored entrainment of locomotor activity by light and temperature in the linden bug Pyrrhocoris apterus (Heteroptera), an insect species with a strong seasonal response (reproductive diapause), which is triggered by both photoperiod and thermoperiod. Our results show that either light or temperature cycles are strong factors entraining P. apterus locomotor activity. Pyrrhocoris is able to be partially synchronized by cycles with temperature amplitude as small as 3°C and more than 50% of bugs is synchronized by 5°C steps. If conflicting zeitgebers are provided, light is the stronger signal. Linden bugs lack light-sensitive (Drosophila-like) cryptochrome. Notably, a high percentage of bugs is rhythmic even in constant light (LL) at intensity ∼400 lux, a condition which induces 100% arrhythmicity in Drosophila. However, the rhythmicity of bugs is still reduced in LL conditions, whereas rhythmicity remains unaffected in constant dark (DD). Interestingly, a similar phenomenon is observed after temperature cycles entrainment. Bugs released to constant thermophase and DD display weak rhythmicity, whereas strong rhythmicity is observed in bugs released to constant cryophase and DD. Our study describes the daily and circadian behavior of the linden bug as a response to photoperiodic and thermoperiodic entraining cues. Although the molecular mechanism of the circadian clock entrainment in the linden bug is virtually unknown, our study contributes to the knowledge of the insect circadian clock features beyond Drosophila research.

• Klíčová slova
• Pyrrhocoris apterus, circadian clock, constant light, entrainment, photoperiod, synchronization, temperature compensation, thermoperiod,
• Publikační typ
• časopisecké články MeSH

Circadian clocks orchestrate daily activity patterns and free running periods of locomotor activity under constant conditions. While the first often depends on temperature, the latter is temperature-compensated over a physiologically relevant range. Here, we explored the locomotor activity of the temperate housefly Musca domestica Under low temperatures, activity was centered round a major and broad afternoon peak, while high temperatures resulted in activity throughout the photophase with a mild midday depression, which was especially pronounced in males exposed to long photoperiods. While period (per) mRNA peaked earlier under low temperatures, no temperature-dependent splicing of the last per 3' end intron was identified. The expression of timeless, vrille, and Par domain protein 1 was also influenced by temperature, each in a different manner. Our data indicated that comparable behavioral trends in daily activity distribution have evolved in Drosophila melanogaster and M. domestica, yet the behaviors of these two species are orchestrated by different molecular mechanisms.

• Klíčová slova
• circadian clock genes, locomotor activity, mRNA splicing, temperature compensation of circadian rhythms, transcription,
• MeSH
• 3' nepřekládaná oblast genetika   MeSH
• časové faktory MeSH
• cirkadiánní rytmus genetika   MeSH
• Drosophila melanogaster genetika   MeSH
• exony genetika   MeSH
• fotoperioda MeSH
• fylogeneze MeSH
• hmyzí geny * MeSH
• introny genetika   MeSH
• kondiční příprava zvířat MeSH
• kryptochromy genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• moucha domácí MeSH
• pohybová aktivita MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese MeSH
• sestřih RNA genetika   MeSH
• teplota * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• kryptochromy MeSH
• messenger RNA MeSH

In temperate regions, the shortening day length informs many insect species to prepare for winter by inducing diapause. The adult diapause of the linden bug, Pyrrhocoris apterus, involves a reproductive arrest accompanied by energy storage, reduction of metabolic needs, and preparation to withstand low temperatures. By contrast, nondiapause animals direct nutrient energy to muscle activity and reproduction. The photoperiod-dependent switch from diapause to reproduction is systemically transmitted throughout the organism by juvenile hormone (JH). Here, we show that, at the organ-autonomous level of the insect gut, the decision between reproduction and diapause relies on an interaction between JH signaling and circadian clock genes acting independently of the daily cycle. The JH receptor Methoprene-tolerant and the circadian proteins Clock and Cycle are all required in the gut to activate the Par domain protein 1 gene during reproduction and to simultaneously suppress a mammalian-type cryptochrome 2 gene that promotes the diapause program. A nonperiodic, organ-autonomous feedback between Par domain protein 1 and Cryptochrome 2 then orchestrates expression of downstream genes that mark the diapause vs. reproductive states of the gut. These results show that hormonal signaling through Methoprene-tolerant and circadian proteins controls gut-specific gene activity that is independent of circadian oscillations but differs between reproductive and diapausing animals.

• MeSH
• cirkadiánní hodiny fyziologie   MeSH
• fotoperioda MeSH
• Heteroptera genetika   metabolismus   MeSH
• hmyzí geny fyziologie   MeSH
• hmyzí proteiny biosyntéza   genetika   MeSH
• kryptochromy biosyntéza   genetika   MeSH
• methopren metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• střevní sliznice metabolismus   MeSH
• transkripční faktory biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• kryptochromy MeSH
• methopren MeSH
• transkripční faktory 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