A transgenerational interval timer inhibits unseasonal sexual morph production in damson-hop aphid, Phorodon humuli

Abstract.  The induction of sexual and parthenogenetic morphs of the damson-hop aphid, Phorodon humuli , on hops is controlled by daylength. The ability of P. humuli , to produce winged pre-sexual females (gynoparae) in the short-day conditions of spring is inhibited by an interval timer present in generations immediately after hatching of the overwintering egg. The inhibition expires after three generations when nymphs are born and reared in short days (LD 12 : 12 h), irrespective of whether their parents are reared in short or long days (LD 18 : 6 h). No gynoparae are produced by aphids maintained for 13 generations in long days. Two wingless aphids from 35 survive transfer from Prunus spinosa to hops. No winged females are produced during nine generations among their progeny maintained in long days on hops, but gynoparae, followed by males, are produced one generation after these aphids are transferred to short days.     

Multigenerational maternal inhibition of prepupal diapause in two Trichogramma species (Hymenoptera: Trichogrammatidae)

It is known that in some insect species the incidence of diapause among the progeny of females that had undergone diapause is relatively low or zero even under strong diapause-inducing conditions. Moreover, the maternal inhibition, preventing the induction of a maladaptive diapause in spring, can persist over several generations. This multigenerational effect based on hypothetical ‘interval timer’ was thoroughly studied in Aphididae. We first described a similar phenomenon in Hymenoptera: laboratory experiments demonstrated that the proportion of diapausing progeny of Trichogramma females that had undergone diapause was practically zero independently of photoperiodic and temperature conditions used (day lengths of 12 and 18 h and temperatures of 12–15 °C). Then the ability to enter diapause recovered gradually and returned to the normal level over two (in Trichogramma telengai) or even five (in Trichogramma principium) generations. We conclude that the observed effect may be based on an interval timer similar to that in aphids.     

Control of the developmental timer forDrosophila pupariation

Summary In the insectDrosophila, formation of the puparium marks the onset of metamorphosis and serves as a useful marker for developmental progress. The cells of the adult remain diploid and divide during the larval stage while the larval cells become polytene and do not divide. We use a high dose of gamma-irradiation (10 krad) to selectively delete the imaginal lineage from the developing larvae ofDrosophila melanogaster. We find that animals depleted of imaginal cells including those of the imaginal brain pupariate only if the larval cells are allowed to mature, demonstrating that the larval cells harbor the primary developmental timer for this process. However, proliferating imaginal cells can exert a negative influence on the timing of pupariation.     

Clock genes period and timeless are rhythmically expressed in brains of newly hatched, photosensitive larvae of the fly, Sarcophaga crassipalpis

While roles of the clock genes period (per) and timeless (tim) are relatively well understood in relation to circadian clocks, their potential roles in insect photoperiodism remain enigmatic. In this study, the expression of per and tim genes under two contrasting photoperiods is described in the central nervous system of photoperiodically sensitive, newly hatched first instar larvae of the flesh fly, Sarcophaga crassipalpis. Using qPCR, diel oscillations were observed in the mRNA levels of both genes under long-day (15 h light:9 h dark, promotes direct development) and short-day conditions (11 h light:13 h dark, induces pupal diapause). Peak per and tim mRNA oscillations were closely associated with the light/dark transition. The conspicuous difference between the two photoperiodic conditions was that the sharp increase in per and tim mRNA abundance occurred during the light phase under long days but during the dark phase under short days. The diel oscillations were, at least in part, driven by an endogenous component, as demonstrated by transferring larvae to continuous darkness. The cells displaying Tim- and Per-like immunoreactivities (Tim- and Per-LIRs) were localized using anti-Drosophila-Per and anti-Chymomyza-Tim antibodies. Per-LIR and Tim-LIR co-localized in three groups of cells in each brain hemisphere. Two other groups, one in the brain hemispheres and the other in the fused ventral nerve ganglion, expressed only the Per-LIR.     

An improved electroantennogram apparatus with a new automatic sample injection system

ABSTRACT. An improved electroantennogram (EAG) apparatus with a new automatic sample injection system, based on a cyclic timer connected to a 12VD.C. electrovalve, is described. The device is fully automatic and allows insertion of constant cyclic air puffs of the test compounds to the antenna at predetermined intervals.     

Accomplishment of time-interval activation of esterase A4 by simple removal of pin fraction

In the seasonal cycle of the silkworm Bombyx mori, an ATPase called esterase A4 (EA4) is thought to measure time interval as a diapause-duration timer. To address the mechanism by which EA4 measures the time, we present a simple EA4 screening method. By the method, EA4 activity can be assessed with a short incubation at 25°C without the need to purify the enzyme further. The method largely overcomes methodological problems that remain unanswered. Besides, the results obtained by the method establish that the time measurement is based on the moment when the time-holding factor(s) PIN (peptidyl-inhibitory needle) is removed. EA4 may originally be a complex with PIN, and external time cues such as winter cold may induce the dissociation of the complex, which in turn results in the timer activation of EA4.     

Dependence of the timing system regulating the onset of gastrulation on cytoplasmic, but not nuclear, activities in the Xenopus embryo

This study examines the properties of the timer that regulates the onset of gastrulation in the Xenopus embryo. Pre-gastrulation embryos were exposed to aphidicolin, vinblastine, 6-dimethylaminopurine (6-DMAP) or urethane. Embryos exposed to aphidicolin or vinblastine for 0.5-2 h before the presumptive onset of gastrulation, began gastrulation at the same time as control embryos. However, those exposed to 6-DMAP or urethane commenced gastrulation significantly later than controls. In 6-DMAP- and urethane-treated embryos, the onset of gastrulation was retarded by approximately 25% and 120%, respectively. 6-DMAP and urethane, but not vinblastine, also lowered the rate of nuclear doubling by 30% and 120%, respectively, in late-blastula to early-gastrula embryos. 6-DMAP and urethane also lowered the rate of cleavage and cleavage-relevant cytoplasmic cycling by 30% and 80%, respectively, in cleavage-stage embryos. We propose that cytoplasmic activities that can be retarded by 6-DMAP and urethane, but not aphidicolin or vinblastine, may be responsible for regulating the onset of gastrulation in Xenopus embryos.     

The Apaf‐1•procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer

During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Tracking protein turnover and degradation by microscopy: photo-switchable versus time-encoded fluorescent proteins

Expanded fluorescent protein techniques employing photo-switchable and fluorescent timer proteins have become important tools in biological research. These tools allow researchers to address a major challenge in cell and developmental biology, namely obtaining kinetic information about the processes that determine the distribution and abundance of proteins in cells and tissues. This knowledge is often essential for the comprehensive understanding of a biological process, and/or required to determine the precise point of interference following an experimental perturbation.     

Delayed setting of the photoperiodic response in recombinant clones of the aphid species Sitobion avenae

1. To explore the possible causes of apparent changes in reproductive mode from obligate to cyclical parthenogenesis over time in recombinant clones of the aphid Sitobion avenae Fabricius, all F1 progenies from various crosses were tested for several consecutive years for sexual morph production, after several weeks' exposure to a short photoperiod. 2. Variable proportions of the F1 progenies from selfing and outcrossing holocyclic clones did not produce mating females when induction was attempted in the year of hatching, but only after further induction, the following year or after. This ‘delayed setting of the photoperiodic response’ (DSPR) was much stronger in recombinants from crosses involving only clones from oceanic regions than in those involving one clone from a region with a continental climate. 3. F1 progenies resulting from crosses between one holocyclic and one intermediate clone did not show DSPR. It appeared again in the F2. 4. DSPR preferentially affected the latest hatched clones in a given progeny. 5. This phenomenon is neither an experimental artefact nor as a result of clone contamination. It appears to be because of a genetically controlled quantitative trait affecting the length of the ‘interval timer’, and may represent an adaptation of holocyclic aphid clones from oceanic regions to unpredictable winter climates.     

An interval timer controls the production of sexual morphs in Myzus persicae (Homoptera: Aphididae)

Abstract The interval timer mechanism was examined in two clones of Myzus persicae (Sulzer) originating from peach in Naousa, North Greece. Overwintering eggs were collected in April 1997 and after egg hatch the morphs of progeny were recorded at LD 10 : 14 h and 17 °C until the appearance of sexuals. In one clone, observations were continued until the 21st generation after egg hatch. In addition, the production of sexual morphs was examined in two other clones originating from Lehonia, Central Greece, when apterae (wingless females) were transferred from long to short days and 17 °C in the second and eighth generation of parthenogenetic rearing after their collection from peach trees. In clones from Naousa, the first gynoparae appear and comprise 10% of alate (winged) females in the second half of the third generation after egg hatch. The percentage of gynoparae increased in each consecutive generation, reaching 100% in fifth and seventh generations in clones Nb and Na, respectively. First males appeared later than gynoparae, in fifth and sixth generation in clones Nb and Na, respectively. In clone Nb, the number of males, after their appearance in fifth generation, was relatively constant from the sixth to the ninth generation (10.8–12.3). Then a significant increase was observed in the 10th generation, thereafter remaining relatively constant until the 21st generation (18.1–19.8). A similar trend in the number of males produced was observed in clones deriving from Lehonia.     

A Natural Fluorescent Protein That Changes Its Fluorescence Color during Maturation

The gene of a new red fluorescent protein zoan2RFP from coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the jellyfish Aequorea victoria, was cloned. At early stages of maturation, zoan2RFP exhibits green fluorescence, which then turns to the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.     

Time-measurement-regulating peptide PIN may alter a timer conformation of Time Interval Measuring Enzyme (TIME)

The TIME (Time Interval Measuring Enzyme) ATPase measures time intervals in accordance with diapause development, which indispensably requires cold for resumption of embryonic development in the silkworm (Bombyx mori). The PIN (Peptidyl Inhibitory Needle) peptide regulates the time measurement function of TIME. In the present study we investigated the interaction between TIME and PIN in order to address the mechanism of diapause development. When TIME was isolated from eggs later than 12 days after oviposition, transient bursts of ATPase activity occurred 18h after isolation of TIME, and the younger the eggs and pupal ovaries from which TIME was isolated, the earlier the bursts of ATPase activity appeared. However, no interval-timer activation of ATPase occurred in ovaries earlier than 6 days after pupation. Similar patterns of ATPase activity occurred in test tubes after mixing TIME with PIN. The shorter the time PIN was mixed with TIME, the earlier the ATPase activity appeared. The timer may be built into the protein conformation of TIME, and PIN (which is present in ovaries beginning 6 days after pupation) appears able to alter this timer conformation through pupal stages to laid eggs. We discuss the possible mechanism of diapause development in relation to the timer mechanism of TIME.     

数字式磁控脊髓夹损仪

在原磁控机械夹断装置的基础上,研制出一种新型的、定量的数字式磁控脊髓夹损仪。该仪器是根据电磁理论和力学平衡原理设计的,由定时器、电流调节器和电流表、控制电路及电磁夹具所组成。电磁夹具左、右两臂间的距离、其单或双臂作用力,运动方向和持续时间均可根据实验要求预先设置和调整。流经电磁夹具单臂或双臂的电流可在数字显示屏上显示并保持。本仪器操作简便可靠,还可直读,适用于复制不同种系动物的不同类型和程度的脊髓损伤。     

The peptide PIN changes the timing of transitory burst activation of timer-ATPase TIME in accordance with diapause development in eggs of the silkworm, Bombyx mori

TIME is an ATPase that measures a time interval by exhibiting transitory burst activation in eggs of the silkworm, Bombyx mori L. PIN is a peptide that regulates the time measurement of TIME. To address the mode of action of PIN, interactions between TIME and PIN were investigated. First, TIME was mixed with PIN for various periods (days) at 25 degrees C. The longer TIME was mixed with PIN, the later the transitory burst activation of TIME ATPase activity occurred, while no such delay occurred at 5 degrees C. Second, the capacity of PIN to bind with TIME was measured at the two temperatures by fluorescence polarization. The binding interaction was much tighter (nearly 1000 times) at 25 degrees C than that at 4 degrees C. Because the log EC50 (in nM) at 4 degrees C was about 7, PIN must dissociate from TIME at low temperatures at the physiological concentration of TIME in eggs. Thus, TIME appears to be restructured into a time-measuring conformation by PIN at the high temperatures of summer, whereas the TIME-PIN complex would dissociate at the low temperatures of winter. This dissociation acts as the preliminary cue for the ATPase activity burst of TIME, which in turn causes the completion of diapause development and initiates new developmental programs.     

Internalization of seasonal time

Orientation of physiology and behavior in time is a major adaptation common to many organisms and represents a significant challenge. In the face of predictable seasonal changes in climatic factors, and as a result of natural selection, many mammals now restrict their reproductive efforts to the fraction of the year when conditions of temperature, food, and water are most favorable for successful weaning of offspring. Changes in day length figure prominently in the synchronization of mammalian seasonal reproductive cycles. In summer breeders (e.g., several hamster, vole, and mouse species), decreasing summer day lengths induce reproductive involution. During this interval, neuroendocrine mechanisms analogous to a simple reference memory permit discrimination of stimulatory from inhibitory photoperiods. The non-reproductive phenotype is sustained for several months thereafter by the inhibitory short days of late summer, autumn, and early winter. Mid-winter reactivation of the reproductive system is triggered after 20-25 weeks of exposure to decreasing or short days by an interval timer that renders the reproductive neuroendocrine system refractory to short days. Recent work that has explored formal and physiological properties of this photorefractoriness interval timer has identified sex differences, neural substrates, and changes in hypothalamic gene expression that may participate in the measurement of seasonal time.