Magdalena Hodkova

Photoperiodic regulation of diapause in linden bugs: are period and Clock genes involved?

Although photoperiodism is undoubtedly one of the most important functions of the circadian system, the role of circadian clock genes remains unclear. We compared the expression of period and Clock genes in the head of the linden bug, Pyrrhocoris apterus, kept under diapause promoting short days (SD) and diapause-preventing long days (LD), using an RNase protection assay. There was only a weak diurnal rhythm in both period and Clock mRNA under LD and no rhythm under SD. Under SD, however, the level of period mRNA was about tenfold and that of clock mRNA about twofold higher than under LD. In a mutant that does not undergo diapause, even under SD, levels of both transcripts were low in both photoperiods. The differential regulation of the levels of two clock gene transcripts in a photoperiodic mutant, demonstrated for the first time in an animal species, strongly indicates a link between photoperiod, the magnitude of clock gene expression, and developmental outputs.

Function of the neuroendocrine complex in diapausing Pyrrhocoris apterus females.

Abstract The role of the brain in inhibiting the action of corpora allata in diapausing short-day females was investigated by transplantation experiments. The function of the transplanted glands was evaluated by oviposition. Active glands from long-day females remained active for a long period of time after transplantation into short-day females, although in situ corpora allata were inhibited shortly after the transfer of females from long to short day. Moreover, inactive glands from short-day females became active after transplantation into other short-day females. In contrast, corpora allata remained inhibited when transplanted together with the brain in the neuroendocrine complex (brain-corpora cardiaca-corpus allatum) where the nervous connections between the brain and corpus allatum remained intact. It is therefore suggested that short-day conditions inhibit corpora allata via nervous connections with the brain.

Seasonal changes in the phospholipid composition in thoracic muscles of a heteropteran, Pyrrhocoris apterus

Abstract The fatty acid composition of phospholipids in thoracic muscles of Pyrrhocoris apterus was related to acclimatization temperature and diapause. Two unsaturated fatty acids, linoleic (18:2n-6) and oleic (18:1n-9), and two saturated, palmitic (16:0) and stearic (18:0), dominated at all temperatures. In contrast to most other reports, the proportion of unsaturated fatty acids did not increase with decreasing temperature; there was a positive correlation between the unsaturation ratio and temperature in total phospholipids ( r =0.67). The most prominent response to cold acclimatization was an increase in the proportion of 16:0 fatty acid and a corresponding decrease in the proportion of fatty acids with 18 carbons. The negative correlation between the proportion of 16:0 and temperature was stronger in phospholipids with phosphatidylethanolamine (PE) head group ( r =−0.85) than in phospholipids with phosphatidylcholine (PC) head group ( r =−0.58). Changes in fatty acid profiles associated with photoperiodic induction of diapause had the same trend as changes related to cold acclimatization. Similar to most other reports, the proportion of PE increased, while the proportion of PC decreased with decreasing temperature. In contrast to a general rule, the PE-phospholipids were less unsaturated than PC-phospholipids.

Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland, corpus allatum

In the conventional view, the winter adaptation of membrane lipids is induced by temperature decrease. We propose that winter remodelling of membranes in Pyrrhocoris apterus is triggered by short-day photoperiod before the temperature decrease and changes caused by cold temperature represent the later phase of adaptation. The induction of diapause by short-day photoperiod results in an accumulation of phosphatidylethanolamine (PE) molecular species with C(16:0)/C(18:2) acyl chains esterified to sn-1/sn-2 positions of glycerol at the expense of C(18:0)/C(18:2). Proportions of C(16:0)/C(18:2)-PE are enhanced in short-day compared to long-day insects in both thoracic muscles (TM, 15.0 vs. 8.2%) and fat bodies (FB, 24.9 vs. 13.6 %). Proportions of C(16:0)/C(18:2)-PE are further enhanced during cold acclimation (to 26.5% in TM, 33.6 % in FB) at the expense of a more saturated species, C(18:0)/C(18:1)-PE. These changes are less prominent in phosphatidylcholines (PC). The effect of photoperiod seems to be mediated via the corpus allatum. Long-day non-diapause females deprived of their corpus allatum have the phospholipid molecular species profile similar to that found in short-day diapausing females. While the acyl chain remodelling is regulated by both photoperiod and temperature, the head group composition is regulated by temperature only. Similar to most other organisms, the level of PE is higher (50.3 vs. 43.5% in TM, 44.3 vs. 37.8% in FB) and that of PC is lower (35.9 vs. 40.2% in TM, 41.6 vs. 46.1 % in FB) at cold temperatures (</=1 degrees C) compared to warm temperatures (>/=16 degrees C). In contrast to a general rule, the PE is less unsaturated than PC. In both TM and FB, proportions of unsaturated/unsaturated molecular species are consistently high in PC (56.3-67.5% in TM, 59.2-66.6% in FB), while they are consistently low in PE (19.1-26.7% in TM, 12.1-15.1% in FB). An adaptive significance of changes in the phospholipid composition for the low temperature and/or dehydration stress is discussed in relation to known physical properties of phospholipids.

Control of diapause and supercooling by the retrocerebral complex in Pyrrhocoris apterus

Effects of external (photoperiod, temperature) and internal (retrocerebral complex, diapause condition) factors on supercooling were studied in Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae) adults. An increase in supercooling capacity is associated with the induction of diapause by short‐day photoperiod or cardiacallatectomy of long‐day non‐diapause insects in spite of a high temperature of 26 °C. The induction of diapause is a prerequisite for a further increase in supercooling capacity by cold acclimation.

Photoperiodic and Food Signals Control Expression Pattern of the Clock Gene, Period, in the Linden Bug, Pyrrhocoris apterus

The temporal expression pattern of the circadian clock gene period was compared between heads of the linden bug, Pyrrhocoris apterus , kept under diapause-promoting short days (SD) and diapause-preventing long days (LD) using a real-time PCR quantification. Diapause or reproduction was programmed by photoperiod during the larval stage, but the first difference in per mRNA abundance between SD and LD insects was observed only after adult ecdysis. The expression level of per mRNA was markedly higher, up to more than 10-fold, in the destined-to diapause animals compared with those scheduled for reproduction. Up-regulation of per transcript was restricted to an early diapause peak, with the maximum expression on days 3 to 5 after adult ecdysis. Starvation reduced the peak level of per mRNA to about 50% of the value found in feeding females in the SD conditions, but per mRNA abundance was similarly low in fasting and feeding females in LD. Photoperiodic refractoriness in either wild-type postdiapause adults or in a selected nondiapause variant of P. apterus was associated with reproduction and low, LD-like levels of per mRNA under both SD and LD. Overall, the data suggest that the photoperiodic programming itself has no direct effect on per mRNA abundance, but it does determine the response of per transcript to food signals during subsequent expression of diapause/reproduction physiology.

Endocrine-dependent expression of circadian clock genes in insects

Abstract.Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tissue of an insect model species, the linden bug Pyrrhocoris apterus, are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the corpus allatum, an endocrine gland producing juvenile hormone. Our results provide the first experimental evidence for the effect of an endocrine gland on circadian clock gene expression in insects.

Effect of thermoperiod on diapause intensity in Pyrrhocoris apterus (Heteroptera Pyrrhocoridae)

The intensity of adult diapause in Pyrrhocoris apterus was measured in two series of experiments as the duration of pre-oviposition period at a constant temperature of 25 degrees C after transfer from short (12L:12D) to long day conditions (18L:6D). Higher diapause intensity was induced with a thermoperiod than at constant temperatures. After the induction throughout larval instars 3-5 and during 4 weeks of adult life at short days and a thermoperiod of 25/15 degrees C the pre-oviposition period was 30+/-4 and 26+/-3 days. After induction at constant 25 degrees C the pre-oviposition period was 22+/-3 and 23+/-4 days, while after induction at constant 20 degrees C it was 17+/-4 and 19+/-4 days. Induction at a lower constant temperature of 20 degrees C was thus followed by a less intense diapause than the induction at a higher constant temperature of 25 degrees C. These counterintuitive results are discussed. The oxygen consumption rate measured at experimental temperatures prior to transfer from short to long days was higher at thermoperiodic conditions than at constant temperatures and it was similar at constant 20 and 25 degrees C. Thus, the oxygen consumption rate measured prior to the transfer was highest (indication of the least intense diapause) in the insects that showed later, after the transfer to long days, the longest pre-oviposition period (indication of the most intense diapause). Within the first two days after transfer to constant 25 degrees C, oxygen consumption rate measured at 25 degrees C decreased in the thermoperiodic insects, while it transiently increased in insects from constant 20 degrees C. Two days and later after the transfer, oxygen consumption rate was similar in all groups. Cold hardiness was not correlated with diapause intensity. The low lethal temperature in diapausing insects was correlated with the night temperature during diapause induction.

Endocrine regulation of non-circadian behavior of circadian genes in insect gut.

The linden bug Pyrrhocoris apterus exhibits a robust diapause response to photoperiod. Photoperiod strongly affected basal levels of circadian gene transcripts in the gut, via the neuroendocrine system. Cryptochrome 2 (cry2) mRNA level was much higher in diapause promoting short days (SD) than in reproduction promoting long days (LD), while Par Domain Protein 1 (Pdp1) mRNA level was higher in LD than in SD. The effect of photoperiod on gene expression was mediated by the neurosecretory cells of the pars intercerebralis (PI) and the juvenile hormone (JH) producing corpus allatum (CA). In LD-females, CA ablation resulted in SD-like levels of gene transcripts, while PI ablation had little effect. Conversely, in SD-females, CA ablation had only a little effect, while PI ablation resulted in LD-like levels of gene transcripts. Thus, the CA is responsible for LD-like characteristics of gene expression in reproducing females and the PI is responsible for SD-like characteristics of gene expression in diapausing females. A simultaneous ablation of both PI and CA revealed two roles of PI in SD-females: (1) inhibition of CA, and (2) weak CA-independent stimulation of cry2 mRNA. Overall, our results indicate that peripheral circadian gene expression in the gut reflects the physiological state of females (with respect to diapause or reproduction) rather than the external light-dark cycle.

Tissue signaling pathways in the regulation of life-span and reproduction in females of the linden bug, Pyrrhocoris apterus.

Molecular studies on Drosophila melanogaster do not provide consistent results with regard to the hormonal regulation of a trade-off between life-span and fecundity. To unravel the physiological basis of the cost of reproduction without affecting animals genotype, a new insect model, Pyrrhocoris apterus, was employed. Reproduction was manipulated by surgical ablations of tissues implicated in reproductive endocrinology, namely the pars intercerebralis (PI) of the brain, the corpus allatum (CA) and the ovary, and the response of life-span to these interventions under diapause-promoting short days and reproduction-promoting long days was measured. Life-span of long-day females increased in the following order: control (high fecundity)=ovary-ablation (no egg production)<PI-ablation (low fecundity)<CA-ablation=ovary+CA-ablation (no reproduction)<CA+PI-ablation (no reproduction). These results show that: (1) PI-signaling (presumably insulin-like peptides) and CA-signaling (juvenile hormone) reduce life-span of long-day females in additive manner and (2) the ovary has no effect on life-span. Life-span of short-day females increased in the following order: PI-ablation (low fecundity)<CA+PI-ablation (no reproduction)<control=CA-ablation (no reproduction). These results implicate factors from the PI that actively extend life-span of short-day females via down-regulation of CA-signaling and also via CA-independent pathway(s). Overall, the data indicate that life-span and reproduction are linked by signals from PI and CA, but, in contrast with the widely held view, neither production of eggs nor gonad-signaling are costly to female longevity.