Weak and strong phase response curves of the onion fly circadian clock at temperature changes of 1 °C and 4 °C.

With increasing soil depth, the amplitude and phase of the daily temperature cycle decreases and is delayed, respectively. The onion fly, Delia antiqua, which pupates at a soil depth of 2-20 cm, advances the eclosion phase of its circadian clock as the temperature amplitude decreases. This "temperature-amplitude response" compensates for the depth-dependent phase delay of the temperature change and ensures eclosion in the early morning. To clarify the physiological mechanisms that induce a temperature-amplitude response, we performed phase-resetting experiments using a 12-h high- or low-temperature pulse with an amplitude of 1 °C or 4 °C. Based on the results obtained, four phase transition curves and four phase response curves were constructed. These curves show that the phase of the eclosion clock shifted more as the magnitude of the temperature change increased. The 24-h temperature cycle delayed, rather than advanced, the phase of the D. antiqua circadian eclosion rhythm. Therefore, we propose that a small phase delay is caused by a small temperature amplitude at a deep site in the soil and a large phase delay is caused by a large temperature amplitude at a shallow site, leading to the temperature-amplitude response exhibited by D. antiqua.

Clock-controlled arylalkylamine N-acetyltransferase (aaNAT) regulates circadian rhythms of locomotor activity in the American cockroach, Periplaneta americana, via melatonin/MT2-like receptor.

Melatonin (MEL) orchestrates daily and seasonal rhythms (eg, locomotion, sleep/wake cycles, and migration among other rhythms) in diverse organisms. We investigated the effects of pharmacological doses (0.03-1 mM) of exogenous MEL intake in the cockroach, Periplaneta americana, on locomotor activity. As per os MEL concentration increased, cockroach locomotor rhythm in light-dark (LD) cycles became more synchronized. The ratio of night activity to 24-h activity increased and the acrophase (peak) slightly advanced. MEL application also influenced total activity bouts in the free-running rhythm. Since MEL slightly influenced τ in the free-running rhythms, it is not a central element of the circadian pacemaker but must influence mutual coupling of multi-oscillatory system components. Arylalkylamine N-acetyltransferase (aaNAT) regulates enzymatic production of MEL. aaNAT activities vary in circadian rhythms, and the immunoreactive aaNAT (aaNAT-ir) is colocalized with the key clock proteins cycle (CYC)-ir and pigment-dispersing factor (PDF)-ir These are elements of the central pacemaker and its output pathway as well as other circadian landmarks such as the anterior and posterior optic commissures (AOC and POC, respectively). It also partially shares immunohistochemical reactivity with PER-ir and DBT-ir neurons. We analyzed the role of Pamericana aaNAT1 (PaaaNAT1) (AB106562.1) by injecting dsRNAaaNAT1 . qPCR showed a decrease in accumulations of mRNAs encoding PaaaNAT1. The injections led to arrhythmicity in LD cycles and the arrhythmicity persisted in constant dark (DD). Continuous administration of MEL resynchronized the rhythm after arrhythmicity was induced by dsRNAaaNAT1 injection, suggesting that PaaaNAT is the key regulator of the circadian system in the cockroach via MEL production. PaaaNAT1 contains putative E-box regions which may explain its tight circadian control. The receptor that mediates MEL function is most likely similar to the mammalian MT2, because injecting the competitive MT2 antagonist luzindole blocked MEL function, and MEL injection after luzindole treatment restored MT function. Human MT2-ir was localized in the circadian neurons in the cockroach brain and subesophageal ganglion. We infer that MEL and its synthesizing enzyme, aaNAT, constitute at least one circadian output pathway of locomotor activity either as a distinct route or in association with PDF system.

Northward expansion of the bivoltine life cycle of the cricket over the last four decades.

Recent climate warming has affected some life-history traits of insects, including voltinism and body size. The magnitude of changes in these traits may differ latitudinally within a species because of the differing lengths of season available for growth. The present study aims to estimate the change in voltinism of the lawn ground cricket, Polionemobius mikado (Shiraki) (Orthoptera: Trigonidiidae), over the last four decades by comparing the body size between adults collected from a wide range of latitudes in Japan in recent years (2015-2017) and those collected four decades ago (1969-1976). The body size of adults collected in recent years showed a latitudinal saw-tooth cline, in the same way as body size did four decades ago, and the cline shifted northward over the last four decades: In 2015-2017, the body size decreased slightly with increasing latitude from 31°N to 36°N, and then increased to 40°N, and again decreased from 40°N to 44°N. Comparison of the body size between recent years and four decades ago revealed that the body size has decreased significantly at the middle latitudes (36-40°N), suggesting that the proportion of smaller bivoltine individuals there has increased over the last four decades. The sum of effective temperatures for postdiapause embryonic development at around 36°N in recent years was comparable to that at 31-35°N four decades ago, at which P. mikado populations were bivoltine. Taken together, these findings suggested that the latitudinal range suitable for the bivoltine life cycle of P. mikado has expanded northward over the last four decades because of climate warming. This is the first report that shows that a decrease in body size can be caused by climate warming via an increase in voltinism.

Day-to-day variations in the amplitude of the soil temperature cycle and impact on adult eclosion timing of the onion fly.

The onion fly Delia antiqua advances its eclosion timing with decreasing temperature amplitude to compensate for a depth-dependent phase delay of the zeitgeber. To elucidate whether or not naturally occurring day-to-day variations in the amplitude of soil temperature cycle disturb this compensatory response, we monitored daily variations in the temperature amplitude in natural soils and evaluated the impact on adult eclosion timing. Our results indicated that both median and variance of the soil temperature amplitude become smaller as depth increases. Insertion of a larger temperature fluctuation into the thermoperiod with smaller temperature amplitude induced a stronger phase delay, while insertion of a smaller temperature fluctuation into the thermoperiod with larger temperature amplitude had a weaker phase-advancing effect. It is therefore expected that larger diurnal temperature fluctuations disturb the compensatory response, particularly if they occur at deeper locations, while smaller temperature fluctuations do so only at shallower locations. Under natural conditions, however, the probability of occurrence of smaller or larger temperature fluctuations in shallower or deeper soils, respectively, is relatively small. Thus, naturally occurring day-to-day variations in the temperature amplitude rarely disturb the compensatory response, thereby having a subtle or negligible impact on adult eclosion timing.

Temperature cycle amplitude alters the adult eclosion time and expression pattern of the circadian clock gene period in the onion fly.

Soil temperature cycles are considered to play an important role in the entrainment of circadian clocks of underground insects. However, because of the low conductivity of soil, temperature cycles are gradually dampened and the phase of the temperature cycle is delayed with increasing soil depth. The onion fly, Delia antiqua, pupates at various soil depths, and its eclosion is timed by a circadian clock. This fly is able to compensate for the depth-dependent phase delay of temperature change by advancing the eclosion time with decreasing amplitude of the temperature cycle. Therefore, pupae can eclose at the appropriate time irrespective of their location at any depth. However, the mechanism that regulates eclosion time in response to temperature amplitude is still unknown. To understand whether this mechanism involves the circadian clock or further downstream physiological processes, we examined the expression patterns of period (per), a circadian clock gene, of D. antiqua under temperature cycles that were square wave cycles of 12-h warm phase (W) and 12-h cool phase (C) with the temperature difference of 8 °C (WC 29:21 °C) and 1 °C (WC 25.5:24.5 °C). The phase of oscillation in per expression was found to commence 3.5h earlier under WC 25.5:24.5 °C as compared to WC 29:21 °C. This difference was in close agreement with the eclosion time difference between the two temperature cycles, suggesting that the mechanism that responds to the temperature amplitude involves the circadian clock.

Brain-midgut cross-talk and autocrine metabolastat via the sNPF/CCAP negative feed-back loop in the American cockroach, Periplaneta americana.

Immunohistochemical reactivities against short neuropeptide F (sNPF-ir) and crustacean cardioactive peptide (CCAP-ir) were detected in both the brain-subesophageal ganglion (Br-SOG) and midgut epithelial cells of the male American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells and decreased the CCAP-ir cells in the Br-SOG, whereas refeeding reversed these effects. The contents of sNPF in the Br-SOG, midgut and hemolymph titer decreased in response to an injection of CCAP into the hemocoel of normally fed male cockroaches, while CCAP titers/contents decreased in response to an injection of sNPF. The results of a double-labeling experiment demonstrated that sNPF-ir co-existed in CCAP-ir cells in the pars intercerebralis (PI), dorsolateral region of protocerebrum (DL), deutocerebrum (De) and SOG. sNPF-ir and CCAP-ir were also colocalized in the midgut. sNPF and CCAP are neuropeptides and midgut factors that interact with each other. Since the two peptides are known to be secreted by identical cells that affect each other, this constitutes autocrine negative feedback regulation for a quick response to food accessibility/inaccessibility. These peptides not only constitute the switch in the digestive mechanism but also couple digestive adaptation with behavior. A CCAP injection suppressed locomotor activity when cockroaches were starved, whereas sNPF activated it when they were fed.

Effects of barley chromosome addition to wheat on behavior and development of Locusta migratoria nymphs.

Locusta migratoria feeds on various Poaceae plants but barley. Barley genes related to feeding deterrence may be useful for developing novel resistant crops. We investigated the effects of barley cultivar Betzes, wheat cultivar Chinese Spring (CS), and six barley chromosome disomic addition lines of wheat (2H-7H) on locomotor activity, feeding behavior, survival and development of L. migratoria nymphs. Locomotor activity was similar in nymphs kept with wheat and 2H-7H in an actograph, whereas it was generally high in those kept with barely. No-choice and choice feeding tests suggested that barley genes related to inhibition of feeding by L. migratoria are located on barley chromosomes 5H and 6H and those related to the palatability of plants on chromosomes 2H, 5H and 6H. Rearing experiments suggested the presence of barley genes negatively affecting the survival and growth of locust nymphs on chromosomes 5H and 2H, respectively, and the effects are phase-dependent.

Phase-dependent locomotor activity in first-stadium nymphs of the desert locust, Schistocerca gregaria: effects of parental and progeny rearing density.

This study examined the effects of parental and progeny rearing density on locomotor activity of 1st-stadium nymphs of the desert locust, Schistocerca gregaria, using an actograph. Progeny obtained from solitarious (isolated-reared) or gregarious (crowd-reared) locusts were reared in isolation or in a group of 30 nymphs. Crowding after hatching had a slight influence on mean activity shortly after the start of measurements, but no clear effect was detected until day 2, when maximum activity during the 6-24 h of observation was significantly higher than that of the nymphs kept in isolation. On the other hand, the effects of parental rearing density on locomotor activity manifested at all ages examined (0-2 days old). Progeny of gregarious locusts showed consistently higher activity than those of solitarious locusts. In newly hatched nymphs, the effect of parental rearing density was explained by variation in body size at hatching, one of the phase-dependent characteristics. Hatchling body color was also correlated with locomotor activity and body weight. Similar levels of locomotor activity were exhibited when green, solitarious and black, gregarious nymphs were similar in body weight. These results suggested that parental rearing density indirectly influences locomotor activity in the progeny shortly after hatching by affecting their body size as eggs or hatchlings.

Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis.

We recorded the eclosion time of the flesh fly, Sarcophaga crassipalpis, at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory, to examine the timing adjustment of eclosion in response to temperature cycles and their amplitudes in the pupal stage. In the soil, most eclosions occurred in the late morning, which was consistent with the eclosion time under pseudo-sinusoidal temperature cycles in the laboratory. The circadian clock controlling eclosion was reset by temperature cycles and free-ran with a period close to 24h. This clock likely helps pupae eclose at an optimal time even when the soil temperature does not show clear daily fluctuations. The eclosion phase of the circadian clock progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased. This response allows pupae located at any depth in the soil to eclose at the appropriate time despite the depth-dependent phase delay of the temperature change. In contrast, the abrupt temperature increase in square-wave temperature cycles reset the phase of the circadian clock to the increasing time, regardless of the temperature amplitude. The rapid temperature increase may act as the late-morning signal for the eclosion clock.

The onion fly modulates the adult eclosion time in response to amplitude of temperature cycle.

To confirm whether the amplitude of diel temperature cycles causes a phase shift of adult eclosion rhythm of the onion fly, Delia antiqua, the peak time (Ø(E)) of adult eclosion was determined under various thermoperiods with a fixed temperature either in the warm or cool phase and temperature differences ranging from 1°C to 4°C between the two phases. Irrespective of the temperature level during the warm or cool phase, Ø(E) occurred earlier with decreasing amplitude of the temperature cycle. The results strongly support the previous conclusion of Tanaka and Watari (Naturwissenschaften 90:76-79, 2003) that D. antiqua responds to the amplitude of temperature cycle as a cue for the circadian adult eclosion timing. The phase advance was larger in thermoperiods with a fixed warm-phase temperature than in those with a fixed cool-phase temperature. This might be ascribed to the interaction between the amplitude and level of temperature in the thermoperiodic regimes.

Effects of parental and progeny rearing densities on locomotor activity of 1st-stadium nymphs in the migratory locust, Locusta migratoria: an analysis by long-term monitoring using an actograph.

The effects of parental and progeny rearing densities on locomotor activity in 1st-stadium nymphs of the migratory locust, Locusta migratoria, were observed over a 24- or 36-h period using an actograph. Newly hatched nymphs showed a small activity peak shortly after hatching and the peak level was significantly higher in offspring (gregarious nymphs) of crowd-reared adults than in those (solitarious nymphs) of isolated-reared adults. However, no significant difference was found between the two groups in maximum activity levels exhibited after the initial peak. Post-hatching crowding enhanced locomotor activity during 2-5h of measurements in 2-day-old nymphs. In this case, the parental density resulted in no significant influence on locomotor activity. However, the maximum activity level shown later in the observation period was higher in gregarious nymphs than in solitarious nymphs. Interestingly, this parental effect was more pronounced in nymphs reared in group than in those reared in isolation. The parental density appeared to affect the degree of response to crowding in the progeny. No evidence was found for the phase accumulation in terms of locomotor activity. The variation observed in locomotor activity among geographical populations did not correspond to their phylogenetic relationships.

Interacting effect of thermoperiod and photoperiod on the eclosion rhythm in the onion fly, Delia antiqua supports the two-oscillator model.

Daily light and temperature cycles entrain adult eclosion rhythms in many insect species, but little is known about their interaction. We studied this problem in the onion fly, Delia antiqua. Pupae were subjected to various combinations of a photoperiod of 12L:12D and thermoperiods. The thermoperiods consisted of 12h warm phase (W) and 12h cool phase (C), giving a mean temperature of 25 degrees C with different temperature steps of 8, 4 and 1 degrees C. As the phase relation of the two Zeitgebers was varied, the phase of eclosion rhythm was shifted, depending on the phase angle with the light cycle and the amplitude of the temperature cycle. When the temperature step in the thermoperiod was 8 degrees C (WC 29:21 degrees C), the eclosion rhythm was entrained mainly to thermoperiod rather than photoperiod. In the regime with a 4 degrees C temperature step (WC 27:23 degrees C), both thermoperiod and photoperiod affected eclosion rhythm, and a phase jump of the eclosion rhythm occurred when the warm phase of thermoperiod was delayed 15-18h from light-on. In regimes with a 1 degrees C temperature step (WC 25.5:24.5 degrees C), the eclosion rhythm was completely entrained to photoperiod. The observed interacting effect of light and temperature cycle on the eclosion rhythm in D. antiqua can be explained by the two-oscillator model proposed by Pittendrigh and Bruce (1959).

The pars intercerebralis as a modulator of locomotor rhythms and feeding in the American cockroach, Periplaneta americana.

It has been shown that in orthopteran insects each of the optic lobes (OLs) contains a circadian pacemaker controlling locomotor activity and that the pars intercerebralis (PI) modifies the activity level. However, the present study showed Period protein-like immunoreactivity (PER-ir) in the PI and dorsolateral protocerebrum (DL) as well as in the OLs in the American cockroach, Periplaneta americana, which raised the possibility that the PI or DL could be a clock element. Therefore, we removed the PI or DL surgically and observed the effects on locomotor rhythms and feeding behavior. In constant darkness (DD), cockroaches with an ablated PI (PIX-DD) showed arrhythmicity in locomotion and a massive increase in food consumption that led to increased body length and weight, while PIX cockroaches reared under LD 12:12 (PIX-LD) and the sham-treated cockroaches in DD (CNT-DD) showed rhythmicity and no increase in food consumption. Statistical analysis showed that arrhythmicity was not accompanied by hyperactivity, suggesting that the PI is involved in the regulation of locomotor activity and feeding in DD. The activities of alpha-amylase and proteases were found to be markedly elevated in the midgut of PIX-DD cockroaches but not in PIX-LD cockroaches. Taken together, these results indicate that the PI modulates locomotor rhythms and feeding behavior of cockroaches in a light-dependent manner. The PI and the OL may regulate circadian rhythms and feeding via distinct pathways.

Adult eclosion timing of the onion fly, Delia antiqua, in response to daily cycles of temperature at different soil depths.

For insects pupating in the soil, the day/night temperature cycle may provide a primary time cue (Zeitgeber) for adult eclosion to occur at an appropriate time of the day. In the soil, however, the phase of temperature cycle is delayed with depth because of the low heat conductivity of the soil. Therefore pupae located deeper in the soil may compensate for the depth-dependent phase delay of Zeitgeber to avoid mistimed emergence. We examined the adult eclosion timing of the onion fly, Delia antiqua, pupating at different depths in soil and under various thermoperiods in the laboratory to determine if such compensation indeed occurs. We found that D. antiqua is able to compensate for the depth-dependent phase delay of the Zeitgeber by advancing the eclosion timing in response to the amplitude of the temperature cycle decreasing with depth.

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua.

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 degrees C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 degrees C, both showed circadian rhythmicity in eclosion. Although the free-running period (tau) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter tau than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in tau.