The formation of a hatching line in the serosal cuticle confers multifaceted adaptive functions on the eggshell of a cicada.

Insect eggshells must meet various demands of developing embryos. These demands sometimes conflict with each other; therefore, there are tradeoffs between eggshell properties, such as robustness and permeability. To meet these conflicting demands, particular eggshell structures have evolved in diverse insect species. Here, we report a rare eggshell structure found in the eggshell of a cicada, Cryptotympana facialis. This species has a prolonged egg period with embryonic diapause and a trait of humidity-inducible hatching, which would impose severe demands on the eggshell. We found that in eggs of this species, unlike many other insect eggs, a dedicated cleavage site, known as a hatching line, was formed not in the chorion but in the serosal cuticle. The hatching line was composed of a fine furrow accompanied by ridges on both sides. This furrow-ridge structure formed in the terminal phase of embryogenesis through the partial degradation of an initially thick and nearly flat cuticle layer. We showed that the permeability of the eggshell was low in the diapause stage, when the cuticle was thick, and increased with degradation of the serosal cuticle. We also demonstrated that the force required to cleave the eggshell was reduced after the formation of the hatching line. These results suggest that the establishment of the hatching line on the serosal cuticle enables flexible modification of eggshell properties during embryogenesis, and we predict that it is an adaptation to maximize the protective role of the shell during the long egg period while reducing the barrier to emerging nymphs at the time of hatching.

Putative Neural Network Within an Olfactory Sensory Unit for Nestmate and Non-nestmate Discrimination in the Japanese Carpenter Ant: The Ultra-structures and Mathematical Simulation.

Ants are known to use a colony-specific blend of cuticular hydrocarbons (CHCs) as a pheromone to discriminate between nestmates and non-nestmates and the CHCs were sensed in the basiconic type of antennal sensilla (S. basiconica). To investigate the functional design of this type of antennal sensilla, we observed the ultra-structures at 2D and 3D in the Japanese carpenter ant, Camponotus japonicus, using a serial block-face scanning electron microscope (SBF-SEM), and conventional and high-voltage transmission electron microscopes. Based on the serial images of 352 cross sections of SBF-SEM, we reconstructed a 3D model of the sensillum revealing that each S. basiconica houses > 100 unbranched dendritic processes, which extend from the same number of olfactory receptor neurons (ORNs). The dendritic processes had characteristic beaded-structures and formed a twisted bundle within the sensillum. At the "beads," the cell membranes of the processes were closely adjacent in the interdigitated profiles, suggesting functional interactions via gap junctions (GJs). Immunohistochemistry with anti-innexin (invertebrate GJ protein) antisera revealed positive labeling in the antennae of C. japonicus. Innexin 3, one of the five antennal innexin subtypes, was detected as a dotted signal within the S. basiconica as a sensory organ for nestmate recognition. These morphological results suggest that ORNs form an electrical network via GJs between dendritic processes. We were unable to functionally certify the electric connections in an olfactory sensory unit comprising such multiple ORNs; however, with the aid of simulation of a mathematical model, we examined the putative function of this novel chemosensory information network, which possibly contributes to the distinct discrimination of colony-specific blends of CHCs or other odor detection.

Anatomical characterization of PDF-tri neurons and peptidergic neurons associated with eclosion behavior in Drosophila.

The peptidergic Pigment-dispersing factor (PDF)-Tri neurons are a group of non-clock neurons that appear transiently around the time of adult ecdysis (=eclosion) in the fruit fly Drosophila melanogaster. This specific developmental pattern points to a function of these neurons in eclosion or other processes that are active around pupal-adult transition. As a first step to understand the role of these neurons, we here characterize the anatomy of the PDF-Tri neurons. In addition, we describe a further set of peptidergic neurons that have been associated with eclosion behavior, eclosion hormone (EH), and crustacean cardioactive peptide (CCAP) neurons, to single cell level in the pharate adult brain. PDF-Tri neurons as well as CCAP neurons co-express a classical transmitter indicated by the occurrence of small clear vesicles in addition to dense-core vesicles containing the peptides. In the tritocerebrum, gnathal ganglion and the superior protocerebrum PDF-Tri neurites contain peptidergic varicosities and both pre- and postsynaptic sites, suggesting that the PDF-Tri neurons represent modulatory rather than pure interneurons that connect the subesophageal zone with the superior protocerebrum. The extensive overlap of PDF-Tri arborizations with neurites of CCAP- and EH-expressing neurons in distinct brain regions provides anatomical evidence for a possible function of the PDF-Tri neurons in eclosion behavior.

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.

Visual and olfactory input segregation in the mushroom body calyces in a basal neopteran, the American cockroach.

The cockroach Periplaneta americana is an evolutionary basal neopteran insect, equipped with one of the largest and most elaborate mushroom bodies among insects. Using intracellular recording and staining in the protocerebrum, we discovered two new types of neurons that receive direct input from the optic lobe in addition to the neuron previously reported. These neurons have dendritic processes in the optic lobe, projection sites in the optic tracts, and send axonal terminals almost exclusively to the innermost layer of the MB calyces (input site of MB). Their responses were excitatory to visual but inhibitory to olfactory stimuli, and weak excitation occurred in response to mechanosensory stimuli to cerci. In contrast, interneurons with dendrites mainly in the antennal lobe projection sites send axon terminals to the middle to outer layers of the calyces. These were excited by various olfactory stimuli and mechanosensory stimuli to the antenna. These results suggest that there is general modality-specific terminal segregation in the MB calyces and that this is an early event in insect evolution. Possible postsynaptic and presynaptic elements of these neurons are discussed.

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster.

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.

Synaptic connections of PDF-immunoreactive lateral neurons projecting to the dorsal protocerebrum of Drosophila melanogaster.

Recent studies in Drosophila melanogaster indicate that the neuropeptide pigment-dispersing factor (PDF) is an important output signal from a set of major clock neurons, s-LN(v)s (small ventral lateral neurons), which transmit the circadian phase to subsets of other clock neurons, DNs (dorsal neurons). Both s-LN(v)s and DNs have fiber projections to the dorsal protocerebrum of the brain, so that this area is a conspicuous locus for coupling between different subsets of clock neurons. To unravel the neural circuits underlying the fly's circadian rhythms, we examined the detailed subcellular morphology of the PDF-positive fibers of the s-LN(v)s in the dorsal protocerebrum, focusing on their synaptic connections, using preembedding immunoelectron microscopy. To examine the distribution of synapses, we also reconstructed the three-dimensional morphology of PDF-positive varicosities from fiber profiles in the dorsal protocerebrum. The varicosities contained large dense-core vesicles (DCVs), and also numerous small clear vesicles, forming divergent output synapses onto unlabeled neurites. The DCVs apparently dock at nonsynaptic sites, suggesting their nonsynaptic release. In addition, a 3D reconstruction revealed the presence of input synapses onto the PDF-positive fibers. These were detected less frequently than output sites. These observations suggest that the PDF-positive clock neurons receive neural inputs directly through synaptic connections in the dorsal protocerebrum, in addition to supplying dual outputs, either synaptic or via paracrine release of the DCV contents, to unidentified target neurons.

Early embryonic development and diapause stage in the band-legged ground cricket Dianemobius nigrofasciatus.

The band-legged ground cricket Dianemobius nigrofasciatus enters diapause at an early embryonic stage when adults are reared under short-day conditions or the eggs are exposed to a low temperature. We examined the morphological features of the embryo during early development and determined the exact stage of entry into diapause. In non-diapause eggs, no periplasmic space was observed in the surface region and a small number of nuclei surrounded by cytoplasm (energids) were found among the yolk granules and lipid droplets 12 h after egg laying (AEL) at 25 degrees C. The energids sparsely but evenly populated the surface region at 40 h AEL, but there were some gaps between these energids. A continuous thin layer of nuclei with cytoplasm had completely covered the egg surface at 56 h AEL, suggesting that the blastoderm is formed between 40 and 56 h AEL. At 72 h AEL, we found a germ band at the posterior pole. Electron microscopy revealed clear cell membranes at 40 h AEL. Staining with rhodamine-dextran dye demonstrated that the cell membrane is formed when the nuclei appear on the egg surface at 12-24 h AEL. These results indicate that cellularization occurs before blastoderm formation. In diapause eggs, neither the embryonic rudiment nor germ band was formed, but a continuous layer of cells covered the egg surface. It is concluded that D. nigrofasciatus enters diapause at the cellular blastoderm.

Synaptic connections between eyelet photoreceptors and pigment dispersing factor-immunoreactive neurons of the blowfly Protophormia terraenovae.

Studies using various mutants of Drosophila melanogaster bearing defects in their visual system, including those of the retinal and extraretinal photoreceptor systems, have indicated that the extraretinal photoreceptor known as the Hofbauer-Buchner (H-B) eyelet plays an active, if subsidiary, role in the entrainment of circadian rhythms. In the present study, in the context of unraveling the function of extraretinal photoreception on circadian rhythms and photoperiodic responses, we searched for extraretinal photoreceptors in the blowfly, Protophormia terraenovae, and found that this fly has a homolog of the H-B eyelet. In addition, we show morphologically direct synaptic connections between the eyelet of P. terraenovae (called here Pt-eyelet, after the species' name) and pigment-dispersing factor (PDF)-immunoreactive neurons, which are putative circadian pacemaker neurons, by immunogold electron microscopy combined with intracellular dye injection. The Pt-eyelet was found to reside in the middle of the posterior surface of the optic lobe between the retina and the lamina, as does the H-B eyelet. This extraretinal photoreceptor was composed of at least four photoreceptor cells equipped with well-organized microvillar rhabdomeres. Rhodopsin 6-like immunoreactivity and also the response to light stimuli clearly showed the Pt-eyelet to be functional. The Pt-eyelet terminals in the accessory medulla exhibited synaptic bouton-like appearances and formed divergent multiple-contact output synapses. Synaptic contacts from the Pt-eyelet terminal to the PDF-immunoreactive neurons were identified by the presence of presynaptic ribbons and accumulated synaptic vesicles. Their possible function is discussed in relation to previous studies on circadian rhythm and photoperiodic response of P. terraenovae.

Synaptic connections between pigment-dispersing factor-immunoreactive neurons and neurons in the pars lateralis of the blow fly Protophormia terraenovae.

In females of the blow fly Protophormia terraenovae, neurons with cell bodies in the pars lateralis (PL) projecting to the retrocerebral complex (designated as PL neurons) are necessary for the induction of reproductive diapause under short-day and low-temperature conditions. In the present study, neural connections between PL neurons and pigment-dispersing factor (PDF)-immunoreactive neurons were examined via immunolight microscopy and immunoelectron microscopy combined with backfills through the cardiac-recurrent nerve. Immunolight microscopy showed that fibers of PL neurons overlapped with PDF-immunoreactive fibers in the dorsolateral region of the superior protocerebral neuropil. Immunoelectron microscopy showed that PDF-immunoreactive fibers formed output synapses with fibers of PL neurons and unlabeled neurons in a region dorsoanteriorly located with respect to the calyx of the mushroom body. The distribution of synaptic connections between PDF-immunoreactive fibers and the fibers of PL neurons was sparse. According to the projection patterns, PDF-immunoreactive fibers with synaptic connections with PL neurons appeared to originate from PDF-immunoreactive neurons with cell bodies at the base of the medulla of the optic lobe (medulla PDF neurons), which are putative circadian clock neurons in P. terraenovae. PDF immunoreactivity was restrictively detected in dense-core vesicles but not in clear synaptic vesicles. The present results suggest that medulla PDF neurons convey time or photoperiodic information to PL neurons for diapause induction through direct synaptic connections.

Synaptic connections of cholinergic antennal lobe relay neurons innervating the lateral horn neuropile in the brain of Drosophila melanogaster.

Presumed cholinergic projection neurons (PNs) in the brain of the fruit fly Drosophila melanogaster, immunoreactive to choline acetyltransferase (ChAT), convey olfactory information between the primary sensory antennal lobe neuropile and the mushroom body calyces, and finally terminate in the lateral horn (LH) neuropile. The texture and synaptic connections of ChAT PNs in the LH and, comparatively, in the smaller mushroom body calyces were investigated by immuno light and electron microscopy. The ChAT PN fibers of the massive inner antennocerebral tract (iACT) extend into all portions of the LH, distributing in a nonrandom fashion. Immunoreactive boutons accumulate in the lateral margins of the LH, whereas the more proximal LH exhibits less intense immunolabeling. Boutons with divergent presynaptic sites, unlabeled as well as ChAT-immunoreactive, appear to be the preponderant mode of synaptic input throughout the LH. Synapses of ChAT-labeled fibers appear predominantly as divergent synaptic boutons (diameters 1-3 microm), connected to unlabeled postsynaptic profiles, or alternatively as a minority of tiny postsynaptic spines (diameters 0.05-0.5 microm) among unlabeled profiles. Together these spines encircle unidentified presynaptic boutons of interneurons which occupy large areas of the LH. Thus, synaptic circuits in the LH differ profoundly from those of the PNs in the mushroom body calyx, where ChAT spines have not been encountered. Synaptic contacts between LH ChAT elements were not observed. The synaptic LH neuropile may serve as an output area for terminals of the ChAT PNs, their presynaptic boutons providing input to noncholinergic relay neurons. The significance of the postsynaptic neurites of the ChAT PNs is discussed; either local or other interneurons might connect the ChAT PNs within the LH, or PNs might receive inputs arising from outside the LH.

The extraretinal eyelet of Drosophila: development, ultrastructure, and putative circadian function.

Circadian rhythms can be entrained by light to follow the daily solar cycle. In Drosophila melanogaster a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Krüppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. We thus demonstrate that eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LN(v)), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues in eyelet. We compared action spectra of entrainment in different fly strains: in flies lacking compound eyes but retaining eyelet (so(1)), lacking both compound eyes and eyelet (so(1);gl(60j)), and retaining eyelet but lacking compound eyes as well as cryptochrome (so(1);cry(b)). Responses to phase shifts suggest that, in the absence of compound eyes, eyelet together with cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring.

Synaptic organization of the mushroom body calyx in Drosophila melanogaster.

The calyx neuropil of the mushroom body in adult Drosophila melanogaster contains three major neuronal elements: extrinsic projection neurons, presumed cholinergic, immunoreactive to choline acetyltransferase (ChAT-ir) and vesicular acetylcholine transporter (VAChT-ir) antisera; presumed gamma-aminobutyric acid (GABA)ergic extrinsic neurons with GABA-like immunoreactivity; and local intrinsic Kenyon cells. The projection neurons connecting the calyx with the antennal lobe via the antennocerebral tract are the only source of cholinergic elements in the calyces. Their terminals establish an array of large boutons 2-7 microm in diameter throughout all calycal subdivisions. The GABA-ir extrinsic neurons, different in origin, form a network of fine fibers and boutons codistributed in all calycal regions with the cholinergic terminals and with tiny profiles, mainly Kenyon cell dendrites. We have investigated the synaptic circuits of these three neuron types using preembedding immuno-electron microscopy. All ChAT/VAChT-ir boutons form divergent synapses upon multitudinous surrounding Kenyon cell dendrites. GABA-ir elements also regularly contribute divergent synaptic input onto these dendrites, as well as occasional inputs to boutons of projection neurons. The same synaptic microcircuits involving these three neuron types are repeatedly established in glomeruli in all calycal regions. Each glomerulus comprises a large cholinergic bouton at its core, encircled by tiny vesicle-free Kenyon cell dendrites as well as by a number of GABAergic terminals. A single dendritic profile may thereby receive synaptic input from both cholinergic and GABAergic elements in close vicinity at presynaptic sites with T-bars typical of fly synapses. ChAT-ir boutons regularly have large extensions of the active zones. Thus, Kenyon cells may receive major excitatory input from cholinergic boutons and considerable postsynaptic inhibition from GABAergic terminals, as well as, more rarely, presynaptic inhibitory signaling. The calycal glomeruli of Drosophila are compared with the cerebellar glomeruli of vertebrates. The cholinergic boutons are the largest identified cholinergic synapses in the Drosophila brain and an eligible prospect for studying the genetic regulation of excitatory presynaptic function.

Neural- and endocrine control of flight muscle degeneration in the adult cricket, Gryllus bimaculatus.

Neural- and endocrine mechanisms controlling degeneration of a dorsal longitudinal flight muscle, M112a, have been studied in adult Gryllus bimaculatus (Orthoptera: Gryllidae). Decapitation completely prevented muscle degeneration. Implantation of a pair of corpora allata or injection of juvenile hormone III into decapitated crickets caused muscle degeneration. Denervation of M112a resulted in reduction of muscle mass compared with that in sham-operated crickets. Denervation of M112a in decapitated crickets, however, did not affect muscle mass. Birefringence and ultrastructure of M112a showed an obvious regional difference in the onset of degeneration. Fibrillar structures of M112a always disappeared from the ventral to dorsal part. Distribution of axon terminals of motor neurons and mechanical responses to the motor nerve stimuli showed that M112a is composed of five motor units with similar twitch properties. When M112a was fully denervated, regional differences in degeneration disappeared. Partial denervation resulted in denervated muscle fibers losing birefringence earlier than innervated fibers. These results suggest that juvenile hormone causes breakdown of flight muscles, and neural factors control degeneration of flight muscles to some extent under the presence of the juvenile hormone.