Clock gene-dependent glutamate dynamics in the bean bug brain regulate photoperiodic reproduction.

Animals adequately modulate their physiological status and behavior according to the season. Many animals sense photoperiod for seasonal adaptation, and the circadian clock is suggested to play an essential role in photoperiodic time measurement. However, circadian clock-driven neural signals in the brain that convey photoperiodic information remain unclear. Here, we focused on brain extracellular dynamics of a classical neurotransmitter glutamate, which is widely used for brain neurotransmission, and analyzed its involvement in photoperiodic responses using the bean bug Riptortus pedestris that shows clear photoperiodism in reproduction. Extracellular glutamate levels in the whole brain were significantly higher under short-day conditions, which cause a reproductive diapause, than those under long-day conditions. The photoperiodic change in glutamate levels was clearly abolished by knockdown of the clock gene period. We also demonstrated that genetic modulation of glutamate dynamics by knockdown of glutamate-metabolizing enzyme genes, glutamate oxaloacetate transaminase (got) and glutamine synthetase (gs), attenuated photoperiodic responses in reproduction. Further, we investigated glutamate-mediated photoperiodic modulations at a cellular level, focusing on the pars intercerebralis (PI) neurons that photoperiodically change their neural activity and promote oviposition. Electrophysiological analyses showed that L-Glutamate acts as an inhibitory signal to PI neurons via glutamate-gated chloride channel (GluCl). Additionally, combination of electrophysiology and genetics revealed that knockdown of got, gs, and glucl disrupted cellular photoperiodic responses of the PI neurons, in addition to reproductive phenotypes. Our results reveal that the extracellular glutamate dynamics are photoperiodically regulated depending on the clock gene and play an essential role in the photoperiodic control of reproduction via inhibitory pathways.

Oviposition-promoting pars intercerebralis neurons show period-dependent photoperiodic changes in their firing activity in the bean bug.

Animals show photoperiodic responses in physiology and behavior to adapt to seasonal changes. Recent genetic analyses have demonstrated the significance of circadian clock genes in these responses. However, the importance of clock genes in photoperiodic responses at the cellular level and the physiological roles of the cellular responses are poorly understood. The bean bug Riptortus pedestris shows a clear photoperiodic response in its reproduction. In the bug, the pars intercerebralis (PI) is an important brain region for promoting oviposition. Here, we analyzed the role of the photoperiodic neuronal response and its relationship with clock genes, focusing on PI neurons. Large PI neurons exhibited photoperiodic firing changes, and high firing activities were primarily found under photoperiodic conditions suitable for oviposition. RNA interference-mediated knockdown of the clock gene period abolished the photoperiodic response in PI neurons, as well as the response in ovarian development. To clarify whether the photoperiodic response in the PI was dependent on ovarian development, we performed an ovariectomy experiment. Ovariectomy did not have significant effects on the firing activity of PI neurons. Finally, we identified the output molecules of the PI neurons and analyzed the relevance of the output signals in oviposition. PI neurons express multiple neuropeptides-insulin-like peptides and diuretic hormone 44-and RNA interference of these neuropeptides reduced oviposition. Our results suggest that oviposition-promoting peptidergic neurons in the PI exhibit a circadian clock-dependent photoperiodic firing response, which contributes to the photoperiodic promotion of oviposition.

Unique morphology and photoperiodically regulated activity of neurosecretory canopy cells in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis exhibits clear photoperiodism in egg laying; it lays more eggs in long-day conditions than in medium-day conditions. A key regulator of egg laying is neurosecretory caudo-dorsal cells (CDCs) producing an ovulation hormone in the cerebral ganglia. Paired small budding structures of the cerebral ganglia (viz. the lateral lobe) also promote egg laying in addition to spermatogenesis and maturation of female accessory sex organs. However, it remains unknown which cells in the lateral lobe are responsible for these. Previous anatomical and physiological studies prompted us to hypothesize that canopy cells in the lateral lobe modulate activity of CDCs. However, double labeling of the canopy cell and CDCs revealed no sign of direct neural connections, suggesting that activity of CDCs is regulated either humorally or through a neural pathway independent of canopy cells. In addition, our detailed anatomical re-evaluation confirmed previous observations that the canopy cell bears fine neurites along the ipsilateral axon and extensions from the plasma membrane of the cell body, although the function of these extensions remains unexplored. Furthermore, comparison of electrophysiological properties between long-day and medium-day conditions indicated that the canopy cell's activity is moderately under photoperiodic regulation: resting membrane potentials of long-day snails are shallower than those of medium-day snails, and spontaneously spiking neurons are only observed in long-day conditions. Thus, canopy cells appear to receive photoperiodic information and regulate photoperiod-dependent phenomena, but not provide direct neural inputs to CDCs.

Neural mechanism of circadian clock-based photoperiodism in insects and snails.

The photoperiodic mechanism distinguishes between long and short days, and the circadian clock system is involved in this process. Although the necessity of circadian clock genes for photoperiodic responses has been demonstrated in many species, how the clock system contributes to photoperiodic mechanisms remains unclear. A comprehensive study, including the functional analysis of relevant genes and physiology of their expressing cells, is necessary to understand the molecular and cellular mechanisms. Since Drosophila melanogaster exhibits a shallow photoperiodism, photoperiodic mechanisms have been studied in non-model species, starting with brain microsurgery and neuroanatomy, followed by genetic manipulation in some insects. Here, we review and discuss the involvement of the circadian clock in photoperiodic mechanisms in terms of neural networks in insects. We also review recent advances in the neural mechanisms underlying photoperiodic responses in insects and snails, and additionally circadian clock systems in snails, whose involvement in photoperiodism has hardly been addressed yet. Brain neurosecretory cells, insulin-like peptide/diuretic hormone44-expressing pars intercerebralis neurones in the bean bug Riptortus pedestris and caudo-dorsal cell hormone-expressing caudo-dorsal cells in the snail Lymnaea stagnalis, both promote egg laying under long days, and their electrical excitability is attenuated under short and medium days, which reduces oviposition. The photoperiodic responses of the pars intercerebralis neurones are mediated by glutamate under the control of the clock gene period. Thus, we are now able to assess the photoperiodic response by neurosecretory cell activity to investigate the upstream mechanisms, that is, the photoperiodic clock and counter.

Phylogenetic Separation of Holotrichia Species (Insecta, Coleoptera, Scarabaeidae) Exhibiting Circadian Rhythm and Circa'bi'dian Rhythm.

A unique two-day rhythm, circabidian rhythm, has been reported in the black chafer, Holotrichia parallela. However, it remains unknown how widely the circabidian rhythm appears in related species. We examined the activity rhythm and phylogeny of congeneric species inhabiting Japan to investigate the appearance of circabidian rhythms in a few subgenera of the genus Holotrichia. We found that Holotrichia picea also exhibited circabidian rhythm. In addition to the regular circabidian pattern, circabidian rhythms with day-switching or with a circadian activity component were also observed. In the day-switching pattern, H. picea switched appearance from odd to even days, or vice versa. In the circadian-like activity patterns, a major night activity and a minor dusk activity appeared alternately. Holotrichia kiotonensis, Holotrichia convexopyga, and Holotrichia loochooana loochooana exhibited a circadian rhythm. Two distinct clades, A and B, were recognized in the histone H3, cytochrome c oxidase subunit 1, and 16S ribosomal RNA phylogenetic trees. This phylogenetic separation was in accordance with the subgeneric classification based on external morphology in a previous study and with behavioral rhythm in the present study: clade A included Nigrotrichia group members, H. kiotonensis, H. convexopyga, H. loochooana loochooana, and H. loochooana okinawana, while clade B included Pedinotrichia group members, H. paralella and H. picea. We suggest that after separation into Nigrotrichia and Pedinotrichia, the behavioral trait of circabidian rhythm probably appeared once in an ancestral species of the Pedinotrichia group, including H. parallela and H. picea.

Mapping PERIOD-immunoreactive cells with neurons relevant to photoperiodic response in the bean bug Riptortus pedestris.

Circadian clock genes are involved in photoperiodic responses in many insects; however, there is a lack of understanding in the neural pathways that process photoperiodic information involving circadian clock cells. PERIOD-immunohistochemistry was conducted in the bean bug Riptortus pedestris to localise clock cells and their anatomical relationship with other brain neurons necessary for the photoperiodic response. PERIOD-immunoreactive cells were found in the six brain regions. In the optic lobe, two cell groups called lateral neuron lateral (LNl) and lateral neuron medial (LNm), were labelled anterior medial to the medulla and lobula, respectively. In the protocerebrum of the central brain, dorsal neuron (Prd), posterior neuron (Prp), and antennal lobe posterior neuron (pAL) were found. In the deutocerebrum, antennal lobe local neurons (ALln) were detected. Double immunohistochemistry revealed that PERIOD and serotonin were not co-localised. Furthermore, pigment-dispersing factor-immunoreactive neurons and anterior lobula neurons essential for R. pedestris photoperiodic response were not PERIOD immunopositive. LNl cells were located in the vicinity of the pigment-dispersing factor immunoreactive cells at the anterior base of the medulla. LNm cells were located close to the somata of the anterior lobula neurons. Fibres from the anterior lobula neurons and pigment-dispersing factor-immunoreactive neurons had contacts at the anterior base of the medulla. It is suggested that LNl cells work as clock cells involved in the photoperiodic response and the region at the medulla anterior base serves as a hub to receive photic and clock information relevant to the photoperiodic clock in R. pedestris.

Photoperiod controls egg laying and caudodorsal cell hormone expression but not gonadal development in the pond snail Lymnaea stagnalis.

Photoperiod is a reliable cue to regulate growth and reproduction for seasonal adaptation. Although photoperiodism has been well studied in Chordata and Arthropoda, less is known about Mollusca. We examined photoperiodic effects on egg laying, body size, gonad-somatic index, oocyte size and relative amounts of caudodorsal cell hormone mRNA in individual rearing conditions in the pond snail Lymnaea stagnalis. Twenty-five weeks after hatching, the percentages of egg-laying snails under a photoperiod of 12 h light and 12 h darkness (12L:12D) were significantly smaller than those under longer days. The total numbers of eggs and egg masses under 12L:12D were significantly smaller than those under longer days. Significant differences between 16L:8D and 12L:12D were not observed in the soft body and ovotestis weight, and the gonad-somatic index. Photoperiodic effects were also not observed in oocyte diameters twenty-two weeks after hatching. Twenty-seven weeks after hatching amounts of caudodorsal cell hormone mRNA were significantly lower in the cerebral ganglia with commissure under 12L:12D than 16L:8D. L. stagnalis exhibited a clear photoperiodic response in egg laying and the amount of caudodorsal cell hormone mRNA, but not in gonadal development. Under 12L:12D suppression of caudodorsal cell hormone expression might suppress egg laying.

Photoperiodic Response in the Pars Intercerebralis Neurons, Including Plast-MIP Neurons, in the Brown-Winged Green Bug, Plautia stali.

Many insects in temperate regions avoid environmental adversity for reproduction, and thus enter reproductive diapause according to photoperiod. This reproductive diapause is induced by inhibition of juvenile hormone biosynthesis in the corpus allatum. Some neuropeptides that have an effect on juvenile hormone biosynthesis have been detected in insect brains. Thus, the reproductive diapause may be photoperiodically regulated by these juvenile hormones-controlling neuropeptides. However, there is limited understanding of how the neurons expressing these neuropeptides respond to the photoperiod and control the peptide release accordingly. Here, we performed electrophysiological analyses in the pars intercerebralis (PI) of Plautia stali, where juvenile hormone inhibitory neuropeptides, Plautia stali myoinhibitory peptides (Plast-MIPs) are expressed. We found that the large neurons in the PI showed very high firing activity under diapause-inducing short day conditions. Neurotracer staining revealed that all recorded neurons projected to the nervus corporis cardiaci 1, which is known to be connected to the corpus cardiacum-corpus allatum complex. Finally, we determined how many of the large PI cells expressed Plast-MIP by single cell reverse transcription PCR. About half of large PI neurons coexpressed Plast-Mip and other neuropeptides, Diuretic hormone 44 and insulin-like peptide 1. The remaining cells only expressed Diuretic hormone 44 and insulin-like peptide 1. The present results suggested that large PI neurons, including Plast-MIP neurons, have enhanced activity under short day conditions, which may increase Plast-MIP release to the corpus cardiacum-corpus allatum complex and thus contribute to reproductive diapause.

Immunoreactive Response of Plast-MIPs to Fasting and Their Functional Role in the Reduction of Hemolymph Reducing Sugars in the Brown-Winged Green Bug, Plautia stali.

Animals survive nutrient deficiency by controlling their physiology, such as sugar metabolism and energy-consuming developmental events. Although research on the insect neural mechanisms of the starvation-induced modulation has progressed, the mechanisms have not been fully understood due to their complexity. Myoinhibitory peptides are known to be neuropeptides involved in various physiological activities, development, and behavior. Here, we analyzed the responsiveness of Plautia stali myoinhibitory peptides (Plast-MIPs) to starvation and their physiological role in the brown-winged green bug, P. stali. First, we performed immunohistochemical analyses to investigate the response of Plast-MIP neurons in the cephalic ganglion to fasting under long day conditions. Fasting significantly enhanced the immunoreactivity to Plast-MIPs in the pars intercerebralis (PI), which is known to be a brain region related to various endocrine regulations. Next, to analyze the physiological role of Plast-MIPs, we performed RNA interference-mediated knockdown of Plast-Mip and injection of synthetic Plast-MIP in normally fed and fasted females. The knockdown of Plast-Mip did not have significant effects on the body weight or proportions of ovarian development in each feeding condition. On the other hand, the knockdown of Plast-Mip increased the gonadosomatic index of normally fed females whereas it did not have a significant effect on food intake. Notably, the knockdown of Plast-Mip diminished the fasting-induced reduction of hemolymph reducing sugar levels. Additionally, injection of synthetic Plast-MIP acutely decreased the hemolymph reducing sugar level. Our results suggested responsiveness of Plast-MIPs in the PI to fasting and their functional role in reduction of the hemolymph reducing sugar level.

The optic lobe-pars intercerebralis axis is involved in circa'bi'dian rhythm of the large black chafer Holotrichia parallela.

The large black chafer Holotrichia parallela exhibits ~ 48-h circa'bi'dian rhythm. Although circabidian rhythm is suggested to involve the circadian clock, no physiological studies have been conducted to verify this involvement. We examined the effects of optic lobe or pars intercerebralis removal on the circabidian rhythm. After removing both optic lobes, all beetles lost their circabidian rhythms (N = 25), but all beetles exhibited circabidian rhythm after removing unilateral optic lobe (N = 18). However, 22% of the latter group exhibited day switching. After removal of the pars intercerebralis, 26.3% beetles showed arrhythmic patterns (N = 19). The number of paraldehyde fuchsin-stained pars intercerebralis cells in the arrhythmic group was significantly reduced compared to in the intact and sham-operated groups. The activity in the pars intercerebralis-removed beetles was significantly higher than that in the control groups. The results show that the optic lobe and at least part of the pars intercerebralis are necessary for circabidian rhythm, and bilateral optic lobes are necessary to maintain regularity of the two-day rhythm in H. parallela. This suggests that a neural circuit of circadian clock cells in the optic lobe to pars lateralis might be evolutionally conserved and used also for the generation of circabidian rhythm.

Immunohistochemical and Direct Mass Spectral Analyses of Plautia stali Myoinhibitory Peptides in the Cephalic Ganglia of the Brown-Winged Green Bug Plautia stali.

For seasonal adaptation, the brown-winged green bug Plautia stali (Hemiptera: Pentatomidae) enters reproductive diapause by suppressing juvenile hormone biosynthesis. Plautia stali myoinhibitory peptides (Plast-MIPs) are known to have allatostatic effects and to suppress juvenile hormone biosynthesis. We examined Plast-MIP-producing neurons in the brain with immunohistochemistry and Fourier transform ion cyclotron resonance mass spectrometry. Rabbit polyclonal antiserum against Plast-MIP revealed immunoreactive cells in seven regions of the brain, including the posterior antennal lobe, basal optic lobe, dorsal anterior protocerebrum, ventrolateral protocerebrum, pars intercerebralis, posterior protocerebrum, and dorsal posterior region to the calyx of the mushroom body, aside from the gnathal ganglion. Anatomical locations of the immunoreactive cells in the pars intercerebralis and dorsal posterior region to the mushroom body calyx partly overlapped with the cell body location stained by retrograde dye fills from the corpus allatum and corpus cardiacum complex. Direct mass spectrometry revealed the molecular ion peaks corresponding to the predictive mass of Plast-MIPs in the pars intercerebralis and the corpus allatum-corpus cardiacum complex. Plast-MIP immunoreactivity in different cell types suggests that Plast-MIPs have different functions in the cephalic ganglia. Considering the anatomical location of neurons projecting to the corpus allatum-corpus cardiacum and results of mass spectrometry, Plast-MIP immunoreactive cells in the pars intercerebralis may play a role in suppressing juvenile hormone biosynthesis.

Juvenile hormone III skipped bisepoxide, not its stereoisomers, as a juvenile hormone of the bean bug Riptortus pedestris.

Juvenile hormone (JH) plays a pivotal role in many aspects of insect physiology. Although its presence was first reported in a blood-sucking bug belonging to the suborder Heteroptera (true bugs), JH species in the group has long been controversial. Although some recent studies proposed a putative JH molecular species in several Heteropteran species, it is not conclusive because physicochemical analyses were insufficient in most cases. Here, we studied this issue with an ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) equipped with C18 and chiral columns in the bean bug Riptortus pedestris (Heteroptera, Alydidae), in which the JH species has long been controversial. Although a recent study describes JHSB3 as the major JH of this species, that finding was not conclusive because its chirality has not been clarified. In the present study, we detected methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named juvenile hormone III skipped bisepoxide (JHSB3), in the culture media of the corpora cardiaca-corpus allatum (CC-CA) complex and in the hemolymph of this species by a chiral ultraperformance liquid chromatography- tandem mass spectrometer (UPLC-MS/MS). Other JHSB3 stereoisomers were not detected. Topical application of JHSB3 effectively averted diapause. These results indicate that JHSB3 is the major JH of R. pedestris. The present study further revealed that JHSB3 and its (2R,3S,10S) isomer are more potent than (2S,3R,10R) and (2S,3R,10S) isomers, which suggests that there is a significance to the configuration of the 2,3-epoxide moiety in JH action. We further found a supplemental significance to the configuration of the 10-position.

Effects of pars intercerebralis removal on circatidal rhythm in the mangrove cricket, Apteronemobius asahinai.

The circatidal rhythm is an endogenous rhythm corresponding to the tidal cycles, and its neural mechanism remains unknown. The mangrove cricket, Apteronemobius asahinai, possesses both circatidal and circadian clocks, and simultaneously exhibits circatidal and circadian rhythms in its locomotor activity. In a previous study, we showed that surgical removal of the optic lobes, the principal circadian clock locus in crickets, disrupted their circadian rhythm, but not their circatidal rhythm. In this study, we focused on the pars intercerebralis (PI) because surgical removal of the PI disrupts the circadian rhythm and causes arrhythmic activity in some cricket species. After surgical removal of the PI, the proportion of crickets displaying circatidal rhythm decreased, and more than half of the crickets exhibited arrhythmic activity. Surgical removal of the regions around the PI also caused a similar effect on locomotor activity. Our results indicate that the PI and/or its surrounding regions are important not only for circadian but also for circatidal rhythm. This suggests the presence of a neural or hormonal pathway in the PI and/or its surrounding regions that is common to the circatidal and circadian rhythms.

Afferent neural pathways from the photoperiodic receptor in the bean bug, Riptortus pedestris.

Adult diapause in the bean bug, Riptortus pedestris, is controlled by the photoperiod, which is received by retinal cells in the central region of the compound eyes. To resolve the afferent neural pathways involved in the photoperiodic response, we examine fibre projections from the photoperiodic receptors to the brain and investigate the roles of the posterior optic tract (POT) in the photoperiodic response. Reduced-silver impregnation and synapsin immunolabelling revealed that the medulla was divided into nine strata: the outer layer comprises 4 strata, the inner layer comprises 4 strata and a serpentine layer separates the inner and outer layers. Biotin injection revealed that retinal fibres from the central region of the compound eye terminated in either the central part of the lamina or the central part of the medulla 3rd or 4th layer. Biotin injection into the central part of the medulla labelled 5 distinct afferent pathways: two terminated in a region of ipsilateral anterior protocerebrum, while the other three had contralateral projections. One pathway ran through the POT and connected to the bilateral medulla serpentine layers. When the POT was surgically severed, diapause incidence under short-day conditions was significantly reduced compared to that observed following a sham operation. However, an incision at a posterior part of the medulla and lobula boundary, as a control experiment, did not affect the photoperiodic response. These results suggest that photoperiodic signals from the central region of the compound eye are transferred to neurons with fibres running in the POT for photoperiodic response in R. pedestris.

Plausible link between circa'bi'dian activity rhythms and circadian clock systems in the large black chafer Holotrichia parallela.

Two-day rhythms, referred to as circa'bi'dian rhythms, have been reported in humans and mosquitos. However, these rhythms only appear under constant conditions, and the functional mechanisms of 2-day rhythms were unknown. Here, we report clear circabidian rhythms of large black chafers (Holotrichia parallela, Coleoptera: Scarabaeidae) in both the laboratory and field. Under 12 h:12 h light:dark (L:D) conditions at 25°C, H. parallela appeared on the ground at the beginning of the dark phase every 2 days. Under constant darkness, H. parallela exhibited free-running with a period of 47.9±0.3 h, suggesting the existence of a clear circabidian rhythm entrained to two 12 h:12 h L:D cycles. Phase responses of the circabidian rhythm to light pulses occurred under constant darkness in a phase-dependent manner. Phase responses suggest that there are two circadian cycles, each consisting of a less-responsive and more-responsive period, in a circabidian oscillation, and the circabidian rhythm is driven by the circadian clock. A mark-recapture study showed that beetles repeatedly appeared on the same tree approximately every 2 days. However, the periodicity was not as rigid as that observed under laboratory conditions in that individuals often switched appearance days. For instance, a large precipitation made the 2-day rhythm shift phase by half a cycle of the rhythm at a time. We propose a novel function of the circadian clock characterized by the release of an output signal every two cycles to produce the 2-day rhythm.

Clock and Hormonal Controls of an Eclosion Gate in the Flesh Fly Sarcophaga crassipalpis.

The eclosion gate in insect development is controlled by the circadian clock and hormonal cascade. To study mechanisms underlying the eclosion gate, we examined eclosion-timing signals from the circadian clock, and the role of 20-hydroxyecdysone in the eclosion gate of the flesh fly, Sarcophaga crassipalpis. Phase responses of the eclosion rhythm were examined by applying a low-temperature pulse in the day prior to the first eclosion peak. A low-temperature pulse applied about 5.4 h before eclosion advanced an eclosion peak by 0.9 h. This indicates that an interval from the Zeitgeber (external environmental cues) input to the behavioral output by the circadian clock is 4.5 h. Signals released by the circadian clock in the last 4.5 h before eclosion could change eclosion time. In the prothoracic gland, daily changes in immunoreactivity against a circadian clock protein PERIOD were observed in the last two days before eclosion. Hemolymph titers of 20-hydroxyecdysone were very low in the last two days of the pupal period. 20-hydroxyecdysone injections caused a delay, not an advancement, in eclosion time in a time dependent manner: pharate adults were sensitive to 20-hydroxyecdysone about 20 and 16 h before eclosion, whereas no significant effects were observed about 12 and 8 h before eclosion. These results suggest that 20-hydroxyecdysone is not a timing signal submitted by the circadian clock but an indicator to suppress premature eclosion. The circadian clock in the prothoracic gland presumably sends a signal distinct from ecdysteroids from several hours before eclosion to time the onset of eclosion.

Neuroanatomy of pars intercerebralis neurons with special reference to their connections with neurons immunoreactive for pigment-dispersing factor in the blow fly Protophormia terraenovae.

Input regions of pars intercerebralis (PI) neurons are examined by confocal and electron microscopies with special reference to their connections with neurons immunoreactive for pigment-dispersing factor (PDF) in the blow fly, Protophormia terraenovae. PI neurons are a prerequisite for ovarian development under long-day conditions. Backfills from the cardiac recurrent nerve after severance of the posterior lateral tracts labeled thin fibers derived from the PI neurons in the superior medial protocerebrum. These PI fibers were mainly synapsin-negative and postsynaptic to unknown varicose profiles containing dense-core vesicles. Backfilled fibers in the periesophageal neuropils, derived from the PI neurons or neurons with somata in the subesophageal zone, were varicose and some were synapsin-positive. Electron microscopy revealed the presence of both presynaptic and postsynaptic sites in backfilled fibers in the periesophageal neuropils. Many PDF-immunoreactive varicosities were found in the superior medial and lateral protocerebrum and double-labeling showed that 60-88 % of PDF-immunoreactive varicosities were also synapsin-immunoreactive. Double-labeling with the backfills and PDF immunocytochemistry showed that the PI fibers and PDF-immunoreactive varicosities were located close to each other in the superior medial protocerebrum. Results of triple-labeling of PI neurons, PDF-immunoreactive neurons and synapsin-immunoreactive terminals demonstrated that the synapsin-positive PDF-immunoreactive varicosities contacted the PI fibers. These data suggest that PI neurons receive synaptic contacts from PDF-immunoreactive fibers, which are derived from circadian clock neurons, of small ventral lateral neurons (previously called OL2) or posterior dorsal (PD) neurons with somata in the pars lateralis.

Both the anterior and posterior eyes function as photoreceptors for photoperiodic termination of diapause in the two-spotted spider mite.

Photoreceptors involved in photoperiodism in insects and mites can be either the retinal photoreceptors in the visual system or nonvisual extraretinal photoreceptors. Mites with no eyes have a clear photoperiodic response, suggesting the involvement of extraretinal photoreceptors in mite photoperiodism. In mites equipped with eyes, however, it is not known whether the retinal or extraretinal photoreceptors are involved in photoperiodism. The two-spotted spider mite Tetranychus urticae possesses two pairs of eyes. Adult females of this species terminate diapause in response to long days. To investigate whether the eyes function as photoperiodic photoreceptors in T. urticae, their eyes were ablated using a laser ablation system. Mites with their eyes intact terminated diapause under long days after low temperature exposure, whereas they remained in diapause under short days. Under constant darkness, they did not terminate diapause. When all eyes were removed, the mites remained in diapause even when they were maintained under long days. In contrast, the mites showed clear photoperiodic response when only the anterior or posterior eyes were removed. These results indicate that both the anterior and posterior eyes function as photoreceptors in photoperiodic termination of diapause in T. urticae.

Involvement of the brain region containing pigment-dispersing factor-immunoreactive neurons in the photoperiodic response of the bean bug, Riptortus pedestris.

The concept of insect photoperiodism based on a circadian clock has been supported by many studies demonstrating that the behavioural circadian rhythm and the photoperiodic response are driven by the same circadian clock genes. However, the neuronal mechanism of the circadian clock underlying photoperiodism is poorly understood. To examine whether circadian rhythm and photoperiodism share a neuronal mechanism, we focused on the neurons that express neuropeptide pigment-dispersing factor (PDF) in the bean bug, Riptortus pedestris. PDF has been identified as an important regulator of the insect circadian rhythm and is expressed in circadian clock neurons of various insect species. In R. pedestris, PDF immunoreactivity was detected in some clusters of cells and their fibres in the optic lobe and the protocerebrum. cDNA encoding a PDF precursor protein was highly conserved between R. pedestris and many other insects. Differences between day and night were not observed in the immunolabelling intensity in cell bodies of PDF-immunoreactive neurons and pdf mRNA expression levels in the head. Surgical removal of the region containing PDF-immunoreactive cell bodies at the medulla disrupted the photoperiodic regulation of diapause. However, gene suppression of pdf by RNA interference did not affect the photoperiodic response. These results suggest that the region containing PDF-immunoreactive somata is important for the photoperiodic response in R. pedestris, but pdf mRNA expression is probably not required for the response.

Significance of the clock gene period in photoperiodism in larval development and production of diapause eggs in the silkworm Bombyx mori.

Many insects living in seasonal environments sense seasonal changes from photoperiod and appropriately regulate their development and physiological activities. Genetic researches have indicated the importance of a circadian clock system in photoperiodic time-measurement for photoperiodic regulations. However, most previous studies have focused on the effects on a single photoperiodic phenotype, without elucidating whether the circadian clock is involved in the core photoperiodic mechanism or only in the production of one target phenotype, such as diapause. Here, we focused on two different phenotypes in a bivoltine Kosetsu strain of the silkworm Bombyx mori, namely, embryonic diapause and larval development, and examined their photoperiodic responses and relationship to the circadian clock gene period. Photoperiod during the larval stage clearly influenced the induction of embryonic diapause and duration of larval development in the Kosetsu strain; short-day exposure leaded to the production of diapause eggs and shortened the larval duration. Genetic knockout of period inhibited the short-day-induced embryonic diapause. Conversely, in the period-knockout silkworms, the larval duration was shortened, but the photoperiodic difference was maintained. In conclusion, our results indicate that the period gene is not causally involved in the photoperiodic response of larval development, while that is essential for the short-day-induced embryonic diapause.