Induction of DNA synthesis by 20-hydroxyecdysone in the prothoracic gland cells of the silkworm Bombyx mori during the last larval instar.

DNA synthesis in the prothoracic gland cells of the silkworm Bombyx mori was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the last larval instar were examined. During the first 3 days of the last larval instar, no DNA-synthesizing cells were detected. On day 4, the number of DNA-synthesizing cells increased and peaked on day 7. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, the number of DNA-synthesizing cells dramatically increased 2 days after 20-hydroxyecdysone treatment and reached a high level on day 5. The mechanism by which 20-hydroxyecdysone treatment induces DNA synthesis of prothoracic gland cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. We conclude that 20-hydroxyecdysone may exert its growth-promoting action indirectly.

Effects of calcium channel antagonists on the corpora allata of adult male loreyi leafworm Mythimna loreyi: juvenile hormone acids release and intracellular calcium level.

The effects of voltage-dependent calcium channel (VDCC) antagonists and the non-specific calcium channel antagonists on both juvenile hormone acids (JHA) release and cytosolic free calcium concentration ([Ca2+]i) are investigated in the corpora allata (CA) of the adult males loreyi leafworm Mythimna loreyi. The VDCC antagonists used in this study are: the L-type antagonists diltiazem, nifedipine, and verapamil, the N-type antagonist omega-Conotoxin (CgTx) GVIA, the N- and P/Q-type antagonist omega-CgTx MVIIC, and the T-type antagonist amiloride. The non-specific calcium channel antagonists used in this study were cadmium (Cd2+), cobalt (Co2+), nickle (Ni2+), and lanthanum (La3+). The results show that both the DHPs-sensitive L-type antagonist nifedipine and the N-type antagonist omega-CgTx GVIA were able to inhibit JHA release, but only omega-CgTx GVIA was able to reduce the [Ca2+]i. Among the non-specific calcium channel antagonists, Cd2+ is the most potent in reducing JHA release but without obvious effect on the [Ca2+]i, La3+ significantly increases the [Ca2+]i but without effect on JHA release.

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori.

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.

Temporal analysis of ecdysteroidogenic activity of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori.

The cellular mechanism underlying ecdysteroidogenesis during the fourth larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid biosynthetic activity of the prothoracic glands, cAMP accumulation of the gland cells, the in vitro release of prothoracicotropic hormone (PTTH), etc. According to the differential responsiveness of prothoracic glands to PTTH, dibutyryl cAMP (dbcAMP), and 1-methyl-3-isobutylxanthine (MIX), the following different stages were classified and changes in PTTH signal transduction were assumed. During the first stage (between days 0 and 1), the glands showed low basal and PTTH-stimulated activities in both cAMP accumulation and ecdysteroidogenesis, and PTTH release in vitro was maintained at low but detectable levels, implying that a low but sustained PTTH signal may be transduced to prothoracic gland cells. On day 1.5, when low basal ecdysteroid production of the prothoracic glands was being maintained, both the responsiveness of glands to the stimulation of PTTH and PTTH release in vitro dramatically increased, indicating greatly increased PTTH transduction. On day 3 (when the basal ecdysteroidogenesis became maximal) and afterwards, high PTTH release in vitro was maintained, but the gland showed no response to PTTH, implying that the refractoriness of gland cells to PTTH may occur at this stage. We assume that the development-specific changes in PTTH signal transduction during the penultimate larval instar may play a critical role in regulating changes in ecdysteroidogenesis of the prothoracic glands.

Mitogenic effects of 20-hydroxyecdysone on neurogenesis in adult mushroom bodies of the cockroach, Diploptera punctata.

The purpose of this study was to examine the mitogenic effects of 20-hydroxyecdysone on neurogenesis in mushroom bodies of the adult cockroach, Diploptera punctata. The occurrence of neurogenesis was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of BrdU-labeled cells in the mushroom bodies was high shortly after adult ecdysis, then gradually decreased, and proliferation ceased on day 8. 20-Hydroxyecdysone injection during the early adult stages significantly delayed the decrease in mitotic activity. Moreover, 20-hydroxyecdysone injection during the late stage stimulated quiescent mushroom body neuroblasts to initiate their mitotic activity in a dose-dependent manner. These results indicated that the mushroom body neuroblasts of this insect become quiescent in the maturing central nervous system, but retain the capacity for proliferation if exposed to appropriate environmental signals. We conclude that 20-hydroxyecdysone has a mitogenic effect on neurogenesis in mushroom bodies of this insect.

Involvement of juvenile hormone in regulation of prothoracicotropic hormone transduction during the early last larval instar of Bombyx mori.

We previously reported that a deficiency in prothoracicotropic hormone (PTTH) transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of corpora allata, as well as larval-pupal transformation. In the present study, the role of juvenile hormone (JH) in the regulation of PTTH transduction was clarified. When JH analog (hydroprene) was applied to early last instar larvae, the development of larvae was greatly inhibited. It was found that it was not PTTH release, but prothoracic gland competency in both cAMP generation and ecdysteroidogenesis to the stimulation of PTTH which was developmentally inhibited by hydroprene application. Glands in hydroprene-treated larvae showed no response in ecdysteroidogenesis to either PTTH or 1-methyl-3-isobutylxanthine (MIX) until day 7, 4 days later than those of control larvae. JH-I application showed the same effects as those of hydroprene. By contrast, allatectomy on day 0 of the last instar accelerated development, and glands showed the activation response to either PTTH or MIX in both cAMP generation and ecdysteroidogenesis 1 day after allatectomy. From these results, we conclude that the absence of JH is a prerequisite for successful PTTH transduction and for acquisition of the cAMP generating system of gland cells.

A deficiency in prothoracicotropic hormone transduction pathway during the early last larval instar of Bombyx mori.

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce ecdysteroids that initiate moulting and metamorphosis. During the last larval instar of holometabolous insects, a reduction in the hemolymph juvenile hormone (JH) levels is a necessary step in initiating larval-pupal transformation. Recently we have demonstrated that very low ecdysteroid levels in the early last larval instar of Bombyx mori initiate the complete inactivation of corpora allata (CA). Results presented here further indicate that PTTH signal transduction pathways undergo specific developmental changes, with a deficiency in transduction in prothoracic gland cells occurring during the early last instar. Glands from the early last instar showed no increase in either cAMP levels or steroidogenesis to the stimulation of PTTH, indicating the absence of the PTTH receptors in gland cells. We propose that this absence of PTTH receptors plays a critical role in directing larval-pupal transformation.

Isolation, identification, and synthesis of sex pheromone components of female tea cluster caterpillar,Andraca bipunctata walker (Lepidoptera: Bombycidae) in Taiwan.

Octadecanal (18:Ald), (E)-11-octadecenal (E11-18:Ald), (E)-14-octadecenal (E14-18:Ald) and (E,E)-11,14-octadecadienal (E11,E14-18:Ald) were isolated and identified as major components from the pheromone glands of the tea cluster caterpillar,Andraca bipunctata, in Taiwan by analyzing the mass spectra of gland components and their DMDS adducts. GC retention times and mass spectra of the components were in agreement with those of authentic synthetic compounds. The average amount of 18:Ald,E11-18:Ald,E14-18:Ald andE11,E14-18:Ald per female gland (1 to 3 days old) was 121±76, 50±20, 187±75, and 237±110 ng, respectively, in a ratio of 20:8:31:41. SyntheticE11,E14-18:Ald caught more males than each of the other three components or blank control in field trapping tests.E11,E14-18:Ald is reported as an insect sex pheromone for the first time. Male antenna responded toE11,E14-18:Ald strongly in an EAG analysis. Furthermore, 4 hr after the injection of PBAN (pheromone biosynthetic activating neuropeptide) into decapitated female moths (2 days old), the percentage of theE11,E14-C18 Ald in the gland extract increased from 0% to 75.5%, which was also significantly more than that of unligated and uninjected control at 55.1%. All these data indicated thatE11,E14-18:Ald is the sex pheromone of theAndraca bipunctata in Taiwan.

Chemical identification of defensive secretion of stick insect,Megacrania tsudai Shiraki.

Volatile constituents of the defensive secretion of the stick insect,Megacrania tsudai Shiraki, in Taiwan have been analyzed by gas chromatography-mass spectrometry (GC-MS). In addition to the major component, actinidine, three more minor components of the secretion are identified as boschniakine and two stereoisomers of 1-acetyl-3-methylcyclopentane by comparing GC retention times with the authentic samples and synthetic compounds. Other components are also tentatively assigned as derivatives of actinidine.

Investigation of sex pheromone components of female asian corn borer,Ostrinia furnacalis (Hübner) (Lepidoptera: Pyralidae) in Taiwan.

(E)-12-Tetradecenyl acetate (E12-14∶OAc), (Z)-12-tetradecenyl acetate (Z12-143nOAc), and tetradecanyl acetate (14∶OAc) were extracted and identified as major chemical components from female tips of the Asian corn borer,Ostrinia furnacalis (Hübner) (Lepidoptera: Pyralidae) in Taiwan, with a combined wash ratio of 48∶37∶15 and an individual female ratio of 45∶39∶16. The average amount ofE12-,Z12-14∶OAc and 14∶OAc in each female gland was 6.6±4.6, 5.8±3.5, and 2.4±1.7 ng/female, respectively. The mixture of these three synthetic chemicals not only gave strong activities in male antennae but also could catch significantly more males than virgin females in field-trapping tests. The field test also showed no significant difference in trapping ability among the three-component Taiwan formulations, the two-component Taiwan formulation (Z12-14∶OAc andZ1214∶OAc, 53∶47), and the Japan formulation (Z12-14∶ OAc andE 12-14∶OAc, 3:2).

Electroantennogram responses of synthetic periplanone-A and periplanone-B in the american cockroach.

The electroantennogram responses of synthetic (-)-periplanone-B (PB) and (-)-periplanone-A (PA) were evaluated by using the antennae of the unmated male American cockroachPeriplaneta americana L. The threshold concentrations of the pheromone activities for the two chemicals were similar. Neither a synergistic nor an inhibitory response was noted, and only an additive effect was detected when PA and PB were applied as a mixture.

Individual variation in sex pheromone of smaller tea tortrix moth,Adoxophyes sp. (Lepidoptera: Tortricidae).

Female smaller tea tortrix mothsAdoxophyes sp. (Lepidoptera: Tortricidae), which initiated calling at 1, 2, or 3 days old, respectively, were analyzed individually for (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (Z)-9-tetradecenyl acetate (Z9-14: OAc) in the pheromone gland via GLC. Among different age groups, broad and similar distributions were found for pheromone quantity (¯X=58.6±52.9 ng/female; range 1.3-219.8 ng/female). The ratio of the two pheromone components averaged 65∶35 but ranged from 84∶16 to 40∶60. The significance of the pheromone blend variation to the attraction of males was tested in a field experiment. The ratio of males trapped by the most attractive blend versus the least attractive one was 2.2.

Evidence for presence and nature of female sex pheromone ofBrithys crini fabricius (Lepidoptera: Noctuidae).

The existence of a female sex pheromone of the noctuid mothBrithys crini Fabricius was confirmed in both laboratory bioassay and field tests. Crude extracts and airborne volatiles from females were analyzed by gas chromatography and mass spectrometry and the data compared with authentic compounds. The primary sex pheromonal compound was Z11-16: Ald. Scanning electron microscopy showed that the external surface of the sex pheromone gland was covered with folds that might increase the sex pheromone evaporation area.

[The application of sex pheromones and juvenile hormone analogs on cockroaches].

The sex pheromone of cockroaches is the chemical which transmits the copulation signal between female and male roaches of the same species. It is produced by virgin females in most roaches, and the chemical structures have been identified for the American, German and Japanese cockroaches. Periplanone-B (PB) and periplanone-A (PA) have been synthesized in the laboratory and tested both in lab and field, showing good biological responses. The sex pheromones of the German cockroach are 3,11-dimethyl-2-nonacosanone, 29-hydroxy-3,11-dimethyl-2-nonacosanone and 29-oxo-3,11-dimethyl-2-nonacosanone, and because of their low volatile nature, they would not be used up in a short time. Periplanone-J (PJ) is produced by the Japanese cockroach. The juvenile hormone analogs (JHA) are chemicals which mimic the juvenile hormones (JH). They have the physiological actions of JH which are maintaining the larvae and nymphs in their immature stages or making the reproductive organs differentiate incompletely, and thereby interfering with their reproduction. The JHA which have been commercialized to control roaches are hydroprene, pyriproxyfen and fenoxycarb. The first two have been proved to reduce the roach population in field tests in the USA and Japan. For some reason, if the sex pheromone and the JHA are used together, the net effect will be more than if they are used alone. In the presence of the sex pheromone, the cockroaches' movements increase, so the chance for a roach to contact the sprayed JHA is also increased. This might be the possible reason for the above observation.

Sex pheromone components of female smaller tea tortrix moth,Adoxophyes sp. (Lepidoptera: Tortricidae) in Taiwan.

(Z)-11-Teiradecenyl acetate (Z11-14: Ac) and (Z)-9-tetrade-cenyl acetate (Z9-14: Ac) were isolated as major sex pheromone components from the female tips of the smaller tea tortrix moth,Adoxophyes sp., in Taiwan. The average amount ofZ11- andZ9-14: Ac in each female gland was 24.9 and 14.1 ng, in a ratio of 64∶36, respectively. When compared to a closely related Japanese strain, which used theZ9-14: Ac,Z11-14∶ Ac,E11-14:Ac, 10-Me-12:Ac four-component system (in a ratio of 63∶31∶4∶2), the Taiwan formulation of two components caught significantly more Taiwan males than the Japanese formulation of four components.

Alarm pheromone of pentatomid bug,Erthesina fullo Thunberg (Hemiptera: Pentatomidae).

In the pentatomid bug,Erthesina fullo Thunberg, the odor of male metathoracic scent gland elicits an alarm response, making the male individuals of the same species alert and disperse; the alarm response of males is more obvious than that of females. Chemical composition of the glandular secretion was identified by gas chromatography and mass spectrometry data in comparison with authentic compounds. No sexual dimorphism exists in the glandular composition in this species. A total of 9 compounds [(E)-2-hexenal, (E)-4-keto-2-hexenal, (E)-2-hexenyl acetate,n-undecane,n-dodecane, (E)-2-decenal,n-tridecane, (E)-2-decenyl acetate, andn-pentadecane] are identified, among whichn-tridecane and (E)-4-keto-2-hexenal comprised nearly 70% of the total secretion in both females and males.

Templeting and self-assembly.

Templeting and self-assembly represent the two extremes of the spectrum of determinate pattern-assembly processes. A templeted pattern can be defined as one that requires a prepattern or templet explicitly specifying the final topology of the pattern. Conversely, a self-assembling pattern can be defined as one for which the inherent constraints of the precursor elements alone are sufficient to specify the final pattern. Both concepts can be directly expressed in matrix notation, and a simple matrix measure, the templeting index, characterizes the relative amount of templeting or of self-assembly in any particular system. With this language, a fundamental principle of pattern-assembly becomes evident: in the determinate realm, some patterns can only be assembled using the same-sized templets--templets that are at least as large as the final pattern.

Attraction responses of the American cockroach to synthetic periplanone-B.

Synthetic periplanone-B has been shown not only to be a sex excitant to malePeriplaneta americana by bioassay in the laboratory but also an attractant pheromone by field tests in a rice storage house in Taipei. During both summer and winter months, it attracted signincantly more adult males into the traps used in the experiments than adult females. There is a statistically significant increase in the sex ratio (male-female) of the trapped adults with increase in periplanone-B used. An attempt has been made to explain the trapping of females and nymphs in addition to males by the chemicals used in our tests.