Sakiko Shiga

Peripheral circadian clock for the cuticle deposition rhythm in Drosophila melanogaster

Insect endocuticle thickens after adult emergence by daily alternating deposition of two chitin layers with different orientation. Although the cuticle deposition rhythm is known to be controlled by a circadian clock in many insects, the site of the driving clock, the photoreceptor for entrainment, and the oscillatory mechanism remain elusive. Here, we show that the cuticle deposition rhythm is regulated by a peripheral oscillator in the epidermis in Drosophila melanogaster. Free-running and entrainment experiments in vitro reveal that the oscillator for the cuticle deposition rhythm is independent of the central clock in the brain driving the locomotor rhythms. The cuticle deposition rhythm is absent in null and dominant-negative mutants of clock genes (i.e., period, timeless, cycle, and Clock), indicating that this oscillator is composed of the same clock genes as the central clock. Entrainment experiments with monochromatic light–dark cycles and cryb flies reveal that a blue light-absorbing photoreceptor, cryptochrome (CRY), acts as a photoreceptor pigment for the entrainment of the cuticle deposition rhythm. Unlike other peripheral rhythms in D. melanogaster, the cuticle deposition rhythm persisted in cryb and cryOUT mutant flies, indicating that CRY does not play a core role in the rhythm generation in the epidermal oscillator.

Roles of PER immunoreactive neurons in circadian rhythms and photoperiodism in the blow fly, Protophormia terraenovae.

SUMMARY Several hypothetical models suggest that the circadian clock system is involved in the photoperiodic clock mechanisms in insects. However, there is no evidence for this at a neuronal level. In the present study, whether circadian clock neurons were involved in photoperiodism was examined by surgical ablation of small area in the brain and by immunocytochemical analysis in the blow fly Protophormia terraenovae. Five types of PER-immunoreactive cells, dorsal lateral neurons (LNd), large ventral lateral neurons (l-LNv), small ventral lateral neurons (s-LNv), lateral dorsal neurons (DNl) and medial dorsal neurons (DNm) were found, corresponding to period-expressing neurons in Drosophila melanogaster. Four l-LNvs and four s-LNvs were bilaterally double-labelled with antisera against pigment-dispersing factor (PDF) and PER. When the anterior base of the medulla in the optic lobe, where PDF-immunoreactive somata (l-LNv and s-LNv) are located, was bilaterally ablated, 55% of flies showed arrhythmic or obscure activity patterns under constant darkness. Percentages of flies exhibiting a rhythmic activity pattern decreased along with the number of small PDF-immunoreactive somata (i.e. s-Lnv). When regions containing small PDF somata (s-LNv) were bilaterally ablated, flies did not discriminate photoperiod, and diapause incidences were 48% under long-day and 55% under short-day conditions. The results suggest that circadian clock neurons, s-LNvs, driving behavioural rhythms might also be involved in photoperiodism, and that circadian behavioural rhythms and photoperiodism share neural elements in their underlying mechanisms.

Neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae.

Abstract. Anatomical study of neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae, was done by NiCl2 filling and immunocytochemistry. Retrograde filling through the cardiac-recurrent nerve labeled three groups of neurons in the brain/subesophageal ganglion: (1) paramedial clusters of the pars intercerebralis, (2) neurons in each pars lateralis, and (3) neurons in the subesophageal ganglion. The pars intercerebralis neurons send prominent axons into the median bundle and exit from the brain via the contralateral nervus corporis cardiaci. Based on the projection pattern, two types of the pars lateralis neurons can be distinguished: the most lateral pairs of neurons contralaterally extend through the posterior lateral tract and the remainder ipsilaterally extend through the posterior lateral tract. The neurons in the subesophageal ganglion run through the contralateral nervus corporis cardiaci. The dendritic arborization of the pars intercerebralis and pars lateralis neurons is restricted to the superior protocerebral neuropil and to the anterior neuropil of the subesophageal ganglion where the neurons in the subesophageal ganglion also project. Retrograde filling from the corpus allatum indicated that the pars lateralis neurons and a few pars intercerebralis neurons project to the corpus allatum, but that the neurons in the subesophageal ganglion do not. Orthograde filling from the pars intercerebralis and staining by paraldehyde-thionin/paraldehyde-fuchsin indicated that the pars intercerebralis neurons project primarily to the corpus cardiacum/hypocerebral ganglion complex. Immunostaining with a polyclonal antiserum against diapause hormone, a member of the FXPRLamide family, suggests that some of the subesophageal ganglion neurons contain FXPRLamide-like peptides.

EFFECTS OF COMPOUND EYE-REMOVAL ON THE PHOTOPERIODIC RESPONSE OF THE BAND-LEGGED GROUND CRICKET, PTERONEMOBIUS NIGROFASCIATUS

The band-legged ground cricket Pteronemobius nigrofasciatus shows a clear photoperiodic response at 25°C with respect to the control of the induction of embryonic diapause. When crickets were reared under a short-day (LD 12 ∶ 12) photoperiod and then transferred to a long-day (LD 16 ∶ 8) photoperiod upon adult emergence, the adults mainly laid nondiapause eggs. However, adults maintained continuously under short-day conditions laid dispause eggs. When compound eyes were bilaterally removed after adult emergence, the crickets mainly laid nondiapause eggs, irrespective of the photoperiod. Thus, the adults completely lost their sensitivity to photoperiod after bilateral removal of their compound eyes. Unilateral removal of the compound eye also affected the crickets under a short-day photoperiod, and the incidence of diapause eggs was intermediate between that laid by intact adults and that laid by adults after the bilateral removal of compound eyes. The incidence of diapause eggs in sham-operated crickets was not significantly different from that in intact crickets under both sets of photoperiodic conditions. These results show that P. nigrofasciatus perceives the photoperiod through its compound eyes.

INDUCTION OF REPRODUCTIVE DIAPAUSE VIA PERCEPTION OF PHOTOPERIOD THROUGH THE COMPOUND EYES IN THE ADULT BLOW FLY, PROTOPHORMIA TERRAENOVAE

Abstract Covering and surgical removal of the compound eyes were performed to localize photoreceptors for photoperiodic responses in the blow fly Protophormia terraenovae (Diptera, Calliphoridae). Adult females showed a long-day photoperiodic response to control reproductive diapause. When the compound eyes were bilaterally covered with silver paint, diapause incidence increased under diapause-preventing conditions, i.e., a long-day photoperiod and constant light, as though flies were kept under constant darkness. Neither silver painting on a medial region of the head capsule nor control painting in which both compound eyes were painted in a clear solvent caused significant effects on diapause incidence. Unilateral painting of the compound eye caused an increment of diapause incidence under constant light but no effects under a long-day photoperiod. When the compound eyes were bilaterally removed, all the flies developed their ovaries both under a long-day and a short-day photoperiod. Unilateral removal of the compound eye also caused ovarian development under a short-day photoperiod, whereas removal of one antennal lobe or all ocelli caused no effects on diapause incidence. Since P. terraenovae completely lost responsiveness to photoperiod after blinding of the compound eyes, it is likely that this fly perceives photoperiod through its compound eyes.

Role of the Median Neurosecretory Cells in the Ovarian Development of the Blow Fly Protophormia terraenovae

Abstract Staining with paraldehyde-thionin-paraldehyde-fuchsin labeled 10–14 median neurosecretory cells (MNC) in each hemisphere of the brain of diapause and nondiapause adults of the blow fly, Protophormia terraenovae (Diptera: Calliphoridae). Various surgical operations were performed on the brain-retrocerebral complex of female adults to investigate the role of these cells in ovarian development and diapause. Under the diapause-averting conditions of LD 18:6 (18 h light and 6 h darkness) and 25°C, most of the intact and sham-operated females had vitellogenic ovaries, whereas all of the females with the MNC completely removed had immature ovaries. The corpus allatum (CA) was significantly smaller in the MNC-removed females than in the intact and sham-operated ones. After severance of the nervi corporis cardiaci, most of the females had immature ovaries. Even after severance of the cardiac recurrent nerve, there were vitellogenic ovaries in about 60 % of the females. However, after removal of the corpus cardiacum and hypocerebral ganglion complex (CCHG), no females had vitellogenic ovaries. Under the diapause-inducing conditions of LD 12:12 and 20°C, most females had immature ovaries and the CA was small irrespective of the surgical procedures. The results indicate that the MNC may secrete an allatotropic factor to stimulate vitellogenesis. The factor seems to be released to the hemolymph from the CCHG and a part of the aorta.

Localization of the Photoreceptor and Pacemaker for the Circadian Activity Rhythm in the Band-Legged Ground Cricket, Dianemobius nigrofasciatus

Abstract Anatomical locations of the photoreceptor and circadian pacemaker for the locomotor activity rhythm were investigated by removal of the external photoreceptors, the compound eyes and ocelli, or the optic lobes in the adult cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae). The activity rhythm of intact crickets freeran under DD with a freerunning period (τ) of 24.6 ± 1.0 h (mean ± SD) and entrained to light-dark cycles of LD 13:13 and LD 12:12. When both compound eyes were removed, some crickets entrained both to LD 13:13 and LD 12:12. Some of the others entrained only to LD 12:12 or showed a complex pattern of the activity. When both compound eyes and all ocelli were removed, some crickets still entrained both to LD 13:13 and LD 12:12. The activity pattern of the crickets receiving a sham operation or unilateral removal of the compound eye was not different from that in the intact crickets. Bilateral removal of the optic lobes caused arrhythmicity both under LD 13:13 and DD, although the activity rhythm in crickets of which the optic lobe was unilaterally removed entrained to LD 13:13 and freeran under DD. These results suggest that D. nigrofasciatus possesses its circadian pacemaker in the optic lobe, and uses both extraretinal photoreceptors and compound eyes for its entrainment.

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.

Juvenile hormone biosynthesis in diapause and nondiapause females of the adult blow fly Protophormia terraenovae.

Abstract In vitro synthetic activities of juvenile hormones (JH) were examined using a radiochemical assay in diapause females and reproductive females of the blow fly, Protophormia terraenovae. Thin layer chromatography showed that products of the corpus allatum (CA) comigrated with a synthetic sample of JH III bisepoxide but neither with JH III nor methylfarnesoate. JH synthetic activities increased in females reared under LD 18:6 at 25°C, as the ovaries developed. The synthetic activities remained low in previtellogenic females reared under LD 12:12 at 20°C. Removal of the pars intercerebralis compelely prevented ovaries from development under reproductive conditions, and removal of the pars lateralis caused partial or full development of ovaries under diapause-inducing conditions. In these operated animals, the JH synthetic activities were not significantly different from those of the intact and sham-operated animals. The results indicate that the CA in P. terraenovae produces mainly JH III bisepoxide and a decrease in the JH production rate is a cause of diapause induction. PI neurons and PL neurons in the brain do not directly mediate changes in the JH production rate, but regulate ovarian development cooperatively with some unknown allatostatic and allatotropic factors.

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly, Protophormia terraenovae

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum-hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg(7)]-corazonin and [Arg(7)]-corazonin(3)(-)(11)) in the PL, 13 peptides (including myosuppressin, [Arg(7)]-corazonin and [Arg(7)]-corazonin(3-11)) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 degrees C) and diapause-averting conditions (LD 18:6 at 25 degrees C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd-10th amino acids were sequenced as -YRKPPFNGS-, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.