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.

A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA-gated chloride channel blockers in the two-spotted spider mite Tetranychus urticae Koch.

The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.

Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori.

Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori.

Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori.

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.

Small GTPases of the Rab family in the brain of Bombyx mori.

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.

Putative regulatory mechanism of prothoracicotropic hormone (PTTH) secretion in the American cockroach, Periplaneta americana as inferred from co-localization of Rab8, PTTH, and protein kinase C in neurosecretory cells.

Small GTPases of the Rab family act as essential regulators of vesicle transport pathways, including the exocytosis of neurohormones. These processes are not well-understood in insects. To address the physiological function of Rab proteins and their phosphorylation in insect neurosecretion, Rab8-like, prothoracicotropic hormone (PTTH)-like, and protein kinase C (PKC)-like immunohistochemical reactivities (-ir) were investigated in the brain of the American cockroach, Periplaneta americana. All the antibodies tested reacted with neurons in the pars intercerebralis, corpora cardiaca, and nervi corporis allati I. Double-labeling experiments demonstrated that all PTTH-ir were colocalized with Rab8-ir and PKC-ir in the pars intercerebralis, although exclusive reactivity was present to antisera against Rab8 or PKC. These findings support the notion that Rab8-like antigen is phosphorylated by PKC, and that this phosphorylation is involved in the axonal transport and secretion of PTTH in this species.

Molecular structure, expression patterns, and localization of the circadian transcription modulator CYCLE in the cricket, Dianemobius nigrofasciatus.

CYCLE (CYC), also known as BMAL1 in vertebrate nomenclature, is a transcription modulator of the circadian genes period and timeless of Drosophila melanogaster. We cloned a cDNA encoding a CYC homologue from the head of the ground cricket, Dianemobius nigrofasciatus (Dncyc), the first CYC from Hemimetabola. The deduced sequence corresponded to a 601 amino-acid polypeptide, with well-defined bHLH, PAS-A, PAS-B, PAC, and BTCR domains. The amino-acid sequence showed 70.7% identity with the CYC protein of Athalia rosae, 63.8% with D. melanogaster, and 52% identity with the human homologue. A cyc transcript of around 3.6kb occurs in the brain, midgut, testis, fatbody, and muscle. An additional band of around 1.1kb gave a hybridization signal in the head. No temporal oscillation in cyc mRNA abundance was observed in the head of the adult cricket when investigated by Northern blot analysis. CYC-like immunohistochemical reactivity (ir) and its dimerization partner CLOCK (CLK)-ir appeared in the pars intercerebralis (PI), tritocerebrum, dorsolateral protocerebrum, and subesophageal ganglion (SOG), but no CYC-ir was observed in the optic lobe (OL) that showed CLK-ir. The deutocerebrum showed a unique CLK-ir but no CYC-ir pattern. Double-labelling experiments showed that both antigens were co-localized in the mandibular and maxillary neuromeres of the SOG. CYC-ir showed no daily oscillation in intensity and the staining pattern was always cytoplasmic. CLK-ir occurred in the nucleus at ZT 16, but was cytoplasmic at other ZT times. A neuronal network equivalent to adult system occurred in the second nymphal stadium.

Molecular characterization and distribution of CYCLE protein from Athalia rosae.

cDNA encoding CYCLE (CYC) from the coleseed sawfly, Athalia rosae (Hymenoptera, Symphyta), was amplified by PCR. This is a first determination of hymenopteran CYC structure. ArCYC had an overall identity of 66% with CYC of Anopheles gambiae and ca. 60% of Drosophila melanogaster. Structural investigation revealed that ArCYC contained characteristic motifs of: bHLH, PAS A, PAS B, PAC and BCTR. Detailed analysis indicated high conservation of these regions among insects. Northern blot analysis showed that the mRNA of ca. 3 kb was transcribed both in the head and in the rest of the body. Southern blot analysis suggested the presence of a single copy of the gene in the genome. Western blot indicates that the quantity of CYC protein does not fluctuate under LD 12:12 in either the head or the rest of the body. Immunocytochemical examination revealed CYC-like antigen in the pars intercerebralis, dorsolateral protocerebrum, dorsal optic tract, tritocerebrum of the brain and the subesophageal ganglion.

Cone Viability Is Affected by Disruption of Melatonin Receptors Signaling.

PURPOSE: Previous studies have demonstrated that melatonin has an important role in the modulation of photoreceptor viability during aging and may be involved in the pathogenesis of age-related macular degeneration.This hormone exerts its influence by binding to G-protein coupled receptors named melatonin receptor 1 (MT1) and 2 (MT2). Melatonin receptors 1 and 2 activate a wide variety of signaling pathways. METHODS: Melatonin-proficient mice (C3H/f+/+) and melatonin-proficient mice lacking MT1 or MT2 receptors (MT1-/- and MT2-/-) were used in this study. Mice were killed at the ages of 3 and 18 months, and photoreceptor viability was determined by counting nuclei number in the outer nuclear layer (ONL). Cones were identified by immunohistochemistry using peanut agglutinin (PNA) and green/red and blue opsin antibodies. Protein kinase B (AKT) and forkhead box O (FOXO1) were assessed by Western blotting and immunohistochemistry. RESULTS: The number of nuclei in the ONL was significantly reduced in C3Hf+/+, MT1-/-, and MT2-/- mice at 18 months of age with respect to 3-month-old animals. In 18-month-old MT1-/- and MT2-/- mice, but not in C3H/f+/+, the number of cones was significantly reduced with respect to young MT1-/- and MT2-/- mice or age-matched C3H/f+/+. In C3H/f+/+, activation of the AKT-FOXO1 pathway in the photoreceptors showed a significant difference between night and day. CONCLUSIONS: Our data indicate that disruption of MT1/MT2 heteromer signaling induces a reduction in the number of photoreceptors during aging and also suggest that the AKT-FOXO1 survival pathway may be involved in the mechanism by which melatonin protects photoreceptors.

Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods.

The evolution of N-acetyltransfeases (NATs) seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT) has been extensively studied since it leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT), and also xenobiotic reactions (arylamine NAT or simply NAT). NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT) form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO) activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the circadian system. Therefore, the enzyme plays both unique and universal roles in insects. The unique role of iaaNATs in physiological regulation urges the targeting of this system for integrated pest management (IPM). We indeed showed a successful example of chemical compound screening with reconstituted enzyme and further attempts seem promising.

N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

Melatonin signaling modulates clock genes expression in the mouse retina.

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT1 and MT2) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.

A novel action of highly specific acaricide; bifenazate as a synergist for a GABA-gated chloride channel of Tetranychus urticae [Acari: Tetranychidae].

Bifenazate is a very selective acaricide that controls the spider mite, Tetranychus urticae. Bifenazate is the first example of a carbazate acaricide. Its mode of action remains unclear. Bifenazate and its active metabolite diazene induce paralysis in spider mites, suggesting that they may act on the nervous system. Here we have employed a homologue (TuGABAR) of RDL (Resistance to dieldrin), a subunit of ionotropic γ-aminobutyric acid (GABA) receptor, from T. urticae to investigate the action of bifenazate and its active metabolite diazene on this receptor function. Although neither acaricide showed a GABA agonist action, 30 µM of bifenazate or diazene significantly enhanced the GABA-induced response of TuGABAR in a dose-dependent manner, shifting the EC(50) of GABA from 24.8 µM to 4.83 µM and 10.8 µM, respectively. This action demonstrates a positive allosteric modulator effect of bifenazate on T. urticae GABA receptors. This synergistic action is likely the result of bifenazate binding to a site distinct from that of the GABA binding site causing a conformational change that affects the magnitude of the GABA response. Precisely how the observed GABA synergist action correlates with the acaricidal activity of bifenazate, if at all, has yet to be determined.

Precursor structure, distribution and possible functions of pigment-dispersing hormone (PDH) in the terrestrial isopod Armadillidium vulgare (Latreille).

Pigment-dispersing hormone (PDH) is an 18 amino acid neuropeptide that induces pigment migration in Decapoda and serves as a circadian neurotransmitter in the locomotor activity rhythm in Drosophila. In this study, a cDNA encoding PDH was cloned from adult brains of the pill bug, Armadillidium vulgare (Av). The cDNA comprising 529 bp encodes a peptide (AvPDH) that consists of a putative 26 amino acid signal peptide, and a 34 amino acid PDH-precursor-related peptide containing an 18 amino acid mature peptide. The peptide shows a high sequence identity (55-77%) to crustacean ß-PDHs and insect PDFs. The tissue-specific expression pattern was examined by reverse transcription PCR. The transcript is expressed in the brain strongly and ventral nerve cord weakly, but the signal was not detected in the intestinal tract. A similar expression profile appeared in Western blot analyses. Western blot analyses with timed samples showed more intense expression of PDH-like antigen at night. PDH-like immunohistochemical reactivity (PDH-ir) was detected in the optic lobe, anteromedian protocerebrum, accessory lobe, tritocerebrum, and suboesophageal ganglion but the reactivity was faint or nil in the pseudofrontal organ (sinus gland). These results were substantiated by in situ hybridization. Co-localization using anti-Gryllus bimaculatus (Gb)-PDF, anti-Bombyx mori (Bm)-CLK, and anti-Bm-CYC showed a co-localization of these antigens in the optic lobe and SOG. The results provide the first structural and immunocytochemical identification of PDH neurons in terrestrial isopods, and the co-localization of PDH with CLK and CYC supports its possible involvement in circadian clock. A day/night rhythm of PDH content is also a new feature.

Co-localization and unique distributions of two clock proteins CYCLE and CLOCK in the cephalic ganglia of the ground cricket, Allonemobius allardi.

CYCLE (CYC) and CLOCK (CLK) are transcriptional activators of the circadian clock genes, period (per) and timeless (tim), binding at E-boxes of their upstream regulatory region in Drosophila. CYC-like and CLK-like immunohistochemical reactivities (CYC-ir and CLK-ir) were investigated in the ground cricket, Allonemobius allardi, in which immunohistochemical reactivities for three circadian clock proteins (PERIOD, Doubletime, and Cryptochrome), two neuropeptides (crustacean cardioactive peptide and diapause hormone), and arylalkylamine-N-acetyltransferase had previously been mapped in the brain-subesophageal ganglion (SOG) complex. CYC-ir and CLK-ir occurred predominantly in the cytoplasm of the neurons distributed mainly in the central brain, SOG, and corpora cardiaca. Double-labeling experiments showed that CYC-ir and CLK-ir were co-localized only in the mandibular and maxillary neuromeres of the SOG. The neuronal processes in the dorsolateral region of the protocerebrum partially shared the immunoreactivities, whereas most of the other immunoreactivities were unique. The optic lobe showed reactivity to anti-CYC at small proximal frontodorsal cells and to anti-CLK at small proximal frontoventral cells. The frontal ganglion exhibited CYC-ir in the cell bodies that lacked CLK-ir. No difference in their number, distribution, or staining intensity was found between sampling under light:dark regimes of 16:8 and 12:12. The levels of both CYC-ir and CLK-ir showed no oscillation throughout a 24-h period. The co-localization pattern suggests that the midline cells of the SOG share most of the circadian-related immunoreactivities, thus constituting the heart of the circadian clock in A. allardi.

Small GTP binding proteins: Rab GTPases from the brain of Bombyx mori.

From a mRNA of the brain of Bombyx mori, we isolated 8 cDNA clones (BRabs), each of which encodes a different member of Rab-protein family. Four of them have more than 80% amino acid identity to the corresponding members of Drosophila Rab proteins. The other 4 proteins show low sequence similarity to any of the known Rab proteins. However, all of them contain the region conserved in rab protein. Using RACE (Rapid Amplification of cDNA ends), the one full-length cDNA clone (BRab14) was isolated. The clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. After purification, the fusion protein was cut with protease to remove GST-Tag and applied to a glutathione S-Sepharose column. The protein bound [(3)H]-GDP with association constant of 1.02 x 10(11) M(-1). Further, the protein was phosphorylated by protein kinase. This result suggests that Rab protein in the brain of Bombyx mori binds GDP or GTP and its function is regulated by phosphorylation.