Akinori Hirashima

Metamorphosis, activity of juvenile-hormone esterase and alteration of ecdysteroid titres: effects of larval density and various stress on the red flour beetle, Tribolium freemani Hinton (Coleoptera: Tenebrionidae).

Abstract When a larva of the red flour beetle Tribolium freemani (Hinton) was introduced into a vial, the larva pupated within 6 days, whereas when two larvae were introduced, the larvae pupated in 12 days. Crowded larvae delayed production of juvenile-hormone esterase (JHE) and release of ecdysteroids, as measured by radioimmunoassay, compared with the isolated larvae. The highest JHE activity was observed at pupal ecdysis in both groups and there were increases in whole-body ecdysteroid levels during the last part of intermoult before pupation and adult emergence, which coincided with decreases in ecdysteroid titres in both isolated and crowded groups. Cold shock, heat stress, the JHE inhibitor methamidophos, and juvenile hormone (JH) I delayed pupation in isolated larvae. Cold shock, heat stress and methamidophos reduced the JHE activity, and cold shock and JH I reduced ecdysteroid levels in isolated larvae. Light, vibration and a nonsteroidal ecdysteroid agonist RH 5849 stimulated pupation in crowded T. freemani larvae, which was antagonized by JH I. The JHE activity and ecdysteroid levels were higher in crowded larvae reared under 24 h light or treated with vibration, octopamine agonists and precocene II than those control larvae kept in constant darkness.

Effect of stress on levels of octopamine, dopamine and serotonin in the American cockroach (Periplaneta Americana L.)

Abstract 1. Various biogenic amines including octopamine, dopamine and serotonin, and their precursors and metabolites in haemolymph and the central nervous system from American cockroaches ( Periplaneta americana L.) were measured using electrochemical detection. 2. Octopamine was found in similar high relative abundances in haemolymph and the central nervous system. 3. The amount of octopamine was much higher than that of tyramine and synephrine in haemolymph and thoracic nerve cord, whereas tyramine was at the highest level followed by octopamine and synephrine in the brain. 4. Insects were stressed by vibrating at 100 or 1000 Hz, visually by flashing light at 4 Hz for 15 min or by immersing the insect in water at 60°C for 30 sec, which resulted in the elevation of octopamine, tyramine, synephrine and tyrosine levels in thoracic nerve cord.

Action of 2-aryliminothiazolidines on octopamine-sensitive adenylate cyclase in the American cockroach nerve cord and on the two-spotted spider mite Tetranychus urticae koch

Abstract The effects of 2-(2,6-diethylphenylimino)thiazolidine (HSO-783) and 2-(4-chloro-2-methylphenylimino)thiazolidine (HSO-786) were compared with those of 2-(2,6-diethylphenylimino)imidazolidine (NC-5) and 2-(4-chloro-2-methylphenylimino)oxazolidine (AC-6) in stimulating adenylate cyclase of Periplaneta americana ventral nerve cord homogenates. These activities were nonadditive with respect to the activity of a maximally effective concentration of octopamine. Washing of homogenates of ventral nerve cord incubated with NC-5, HSO-783, AC-6, and HSO-786 removed nearly all of the stimulatory activities of these agonists. Maximal stimulation of nerve cord adenylate cyclase activity by NC-5, HSO-783, AC-6, and HSO-786 was inhibited by several antagonists, including mianserin, cyproheptadine, chloromazine, and gramine. The rank-order ability of these antagonists to block the maximal adenylate cyclase activation by NC-5, HSO-783, AC-6, and HSO-786 was identical to the rank-order ability of the same antagonists to block the enzyme activation by an optimally effective concentration of octopamine. The β-adrenergic antagonist propranolol was less potent in this respect. AC-6 was a much better acaricide than HSO-783, HSO-786, and NC-5, which were much less potent octopaminergic agonists than AC-6. HSO-786 was a more potent acaricide and octopaminergic agonist than HSO-783. These observations suggest that the toxicity of NC-5, HSO-783, AC-6, and HSO-786 may be due to their octopaminergic agonist action.

Effects of various stressors on larval growth and whole-body octopamine levels of Tribolium castaneum

Abstract Dietary chemical stressors including insecticidal acetylcholinesterase (AChE)-inhibiting organophosphorus compounds and carbamate induced an increase of whole-body octopamine levels and a reduction of weight gain of Tribolium castaneum larvae 72 hr after treatment. Organophosphorus compounds with very poor or no anti-AChE nor insecticidal activities did not have any significant effects, suggesting that the inhibition of AChE may be responsible for these biological phenomena. Rotenone, ethofenprox, nereistoxin, lindane, mechanical stress (40 rpm), high temperature (40°C), and starvation for 48 hr were also effective in reducing larval growth and increasing the whole-body octopamine level of T. castaneum . Social stress, nicotine, and low temperature (20°C) diminished whole-body octopamine levels, whereas allethrin and KK-42 increased the octopamine levels without affecting the larval growth. There seems to be a correlation between increased whole-body octopamine levels and reduced larval-weight gain, indicating that various stressors may play an important role in regulating insect growth, in which octopamine is involved.

Titres of biogenic amines and ecdysteroids: effect of octopamine on the production of ecdysteroids in the silkworm Bombyx mori

At day two, a sharp peak of octopamine (OA) was observed in last instar female Bombyx mori larvae. This peak also appeared in male larvae a day later than in females at day three. An OA peak was also observed before the 3rd ecdysis. However, no OA peaks were observed in 4th instar larvae. At day eight and nine of the 5th instar, another OA peak was observed for male and female, respectively. A peak of tyramine (TA) was found at day one followed by a peak of OA at day two in 3rd instar larvae. At day two, a day before OA peak, a peak of TA was observed for male insects and before the 2nd peak of OA, TA titre was also high in 5th instar larvae. Immediately after 3rd ecdysis, a high titre of DL-beta-(3,4-dihydroxyphenyl)alanine (DOPA) was observed, followed by a peak of dopamine (DA) at day five. A peak of DOPA was found at day one followed by a peak of DA at day two in 3rd instar larvae. Similarly, a small peak of DOPA was observed at day two, followed by an increase of DA at days eight and nine after the 4th ecdysis. Ecdysteroid peaks were observed just before the 3rd and 4th ecdysis and an ecdysteroid titre increased after the start of spinning. The effects of OA and JH on production of ecdysteroids by prothoracic glands (PGs) were examined in order to identify neuromediators responsible for triggering pupation in B. mori larvae. Exogeneous OA (10-100 mM) reduced and 10 &mgr;M OA stimulated the production of ecdysteroids in the presence and absence of brain extracts by PGs in the final instar (day five) of B. mori in vitro. Meanwhile, exogeneous JHI (10 &mgr;g/ml) stimulated and at 5 &mgr;g/ml it reduced production of ecdysteroids in the presence of brain extracts. Gramine, an OA antagonist, delayed pupation when applied in the diet. Thus, OA may produce some biological effects on the programming of larval-pupal development.

Synthesis and Octopaminergic Agonist Activity of 2-(Substituted benzylamino)-2-thiazolines

2-(Substituted benzylamino)-2-thiazolines (SBAT) were synthesized by a hydrochloric acid-catalyzed cyclization of the corresponding thioureas, using a reaction of 2-methylthio-2-thiazoline with substituted benzylamines or by alkylating 2-amino-2-thiazoline. 2-(Alkylthio)-2-thiazolines were obtained by alkylating 2-mercaptothiazoline. Most of the SBAT compounds activated adenylate cyclase in homogenates of cockroach ventral nerve cords; the effect of introducing substituents at the phenyl of the SBAT compounds on octopaminergic agonist activity was not significant. 2-[β-(Substituted phenyl)ethylamino]-2-thiazolines and 2-(alkylthio)-2-thiazolines were not significant octopaminergic agonists. Washing removed nearly all of the activity of one of the SBAT compounds, suggesting that the SBAT compounds bound reversibly to the octopaminergic receptor.

Homology modeling, agonist binding site identification, and docking in octopamine receptor of Periplaneta americana

AY333178 (from Periplaneta americana, 628 AAs) was selected as a target octopamine receptor (OAR) class OAR2 for this study using Discovery Studio (DS Modeling1.1/1.2, Accelrys Inc.). Blast similarity search was performed and identified that AY333178 contains N-terminal domain of GPCR. Based upon Blast and Pfam results, Rhodopsin 1U19 (protein data bank) was considered as an ideal homologue and used as a template for homology modeling due to its higher X-ray resolution at 2.2A. Sequence alignment between AY333178 and 1U19 was done using Align123 followed by a manual modification. The final alignment was carefully evaluated and evidenced to be matching the conserved residue data for class A GPCR fairly well. The 3D model of AY333178 was generated with MODELER, and further refined using CHARMm. Superimposition of the model was done over the template 1U19. Two fairly consistent profiles were observed demonstrating AY333178 model was reasonable and could be employed for the further docking study. Agonist docking into OAR2 model was done using LigandFit. The superimposition of two top poses of representative agonists was performed with a soft surface generated. Those models are considered to be used in designing new leads for hopefully more active compounds. Further research on the comparison of models for the agonists may elucidate the mechanisms of OAR2-ligand interactions.

Three-dimensional common-feature hypotheses for octopamine agonist 2-(arylimino)imidazolidines

Three-dimensional pharmacophore hypotheses were built from a set of 10 octopamine (OA) agonist 2-(Arylimino)imidazolidines (AIIs), 2-(Arylimino)thiazolidines (AITs) and 2-(Arylimino)oxazolidines (AIOs). Among the 10 common-featured models generated by program Catalyst/HipHop, a hypothesis including a ring aromatic (RA), a positive ionizable (PI) and three hydrophobic aliphatic (HpAl) features was considered to be important in evaluating the OA-agonist activity. Active OA agonist 2,6-Et2 AII mapped well onto all the RA, PI and HpAl features of the hypothesis. On the other hand, less active compounds were shown to be difficult to achieve the energetically favorable conformation which is found in the active molecules in order to fit the 3-D common-feature pharmacophore models. Taken together, 2,6-Et2-Ph and foramidine structures are important as OA agonists. The present studies on OA agonists demonstrate that a RA, a PI and three HpAl sites located on the molecule seem to be essential for OA-agonist activity.

Role of the ecdysteroid system in the regulation of Drosophila reproduction under environmental stress.

In a normal environment, both ecdysteroids and the juvenile hormone are known to control reproduction in Drosophila . These hormones normally initiate the synthesis of vitelline proteins in the fat body immediately after emergence and sustain it at a certain level in fertilized flies, and regulate the absorption of vitelline proteins by oocytes [1–4]. However, little is known about the ecdysteroid system in mature Drosophila exposed to stress. To our knowledge, only one report dealt with this problem; an increased level of 20-hydroxyecdysone was found in whole bodies of starving D. melanogaster females (the level of ecdysone was not determined) [2]. We previously revealed considerable changes in the Drosophila reproductive function under stress [5] and suggested that the ecdysteroid system (ecdysone and 20-hydroxyecdysone) was involved in these events.

Quantitative structure-activity studies of octopaminergic 2-(arylimino)thiazolidines and oxazolidines against the nervous system of Periplaneta americana L.

The quantitative structure-activity relationship (QSAR) of octopaminergic 2-(arylimino)thiazolidines (AITs) and 2-(arylimino)oxazolidines (AIOs) against the thoracic nerve cord of the American cockroach, Periplaneta americana L., was analysed using reported physicochemical parameters and regression analysis. The more electron-donating, the less bulky at m-position, and the more hydrophobic the substituent, the greater the activity. The plots of observed log Vmax values against calculated log Vmax values having substituents on the m-position deviated downwards from those of compounds having substituents at the 0- and/or p-positions. The more hydrophobic and the more electron-withdrawing the substituent, the greater the activity. AIO with a 2, 3, 4-trichlorophenyl group (58) was more active than its thiazolidine derivative, 2-(2,3,4-trichlorophenylimino)thiazolidine (38) in terms of Vmax:Vmax of 58 was 30% relative to octopamine (OA), whereas that of 38 has been 9% relative to OA, respectively. Superimposition of energy-minimized OA and 58 revealed structural and conformational similarities that might account for the high activity of 58.