Precocenes as pro-allatocidins in adult female Diploptera punctata: A functional and ultrastructural study

Adult mated females of the viviparous cockroach Diploptera punctata are moderately sensitive to precocenes. Oöcyte growth is inhibited and oviposition is delayed in insects topically treated with precocene II or precocene III. C₁₆ juvenile hormone release by corpora allata of precocene-treated insects is markedly inhibited when compared to corpora allata of acetone-treated controls. Electron microscopy of the corpora allata reveals that precocene treatment results in a disorganisation of the intracellular organelles. Topically applied precocene II reaches a high concentration in the haemolymph (0.5 mM 2 hr after topical application of 250 μg). C₁₆ juvenile hormone release by isolated corpora allata is inhibited by precocenes in vitro; half-maximal inhibition over a 3 hr period is obtained at 0.4 mM precocene II. In vitro inhibition of corpora allata by precocene II concentrations higher than 1 mM rapidly destroys the glands as evidenced by electron microscopy (total disintegration of cellular organelles) and by the virtual cessation of C₁₆ juvenile hormone synthesis by the corpora allata. Inhibition of C₁₆ juvenile hormone release by precocene is time-dependent and is not reversible over the short-term incubation in vitro. This inhibition does not appear to be related to the spontaneous activity of the glands in vitro, and it can be reduced by two epoxidase inhibitors. Precocenes are pro-allatocidins in this species: they are bioactivated within the corpora allata to cytotoxic epoxides.     

Precocene II,a Trichothecene Production Inhibitor,Binds to Voltage-Dependent Anion Channel and Increases the Superoxide Level in Mitochondria of Fusarium graminearum

Precocene II, a constituent of essential oils, shows antijuvenile hormone activity in insects and inhibits trichothecene production in fungi. We investigated the molecular mechanism by which precocene II inhibits trichothecene production in Fusarium graminearum, the main causal agent of Fusarium head blight and trichothecene contamination in grains. Voltage-dependent anion channel (VDAC), a mitochondrial outer membrane protein, was identified as the precocene II-binding protein by an affinity magnetic bead method. Precocene II increased the superoxide level in mitochondria as well as the amount of oxidized mitochondrial proteins. Ascorbic acid, glutathione, and α-tocopherol promoted trichothecene production by the fungus. These antioxidants compensated for the inhibitory activity of precocene II on trichothecene production. These results suggest that the binding of precocene II to VDAC may cause high superoxide levels in mitochondria, which leads to stopping of trichothecene production.     

Precocene I and II inhibition of vitellogenic oöcyte development in Drosophila melanogaster

Adult female Drosophila melanogaster were exposed to precocene I and II, antiallatropin compounds which result in juvenile hormone deficiency in many insects. The presence of juvenile hormone in Drosophila adults was evaluated by examining vitellogenic oöcyte development, a process regulated by juvenile hormone in these flies. Both precocenes reduced the number of vitellogenic oöcytes present 43 hr after exposure in a dose-dependent manner. Precocene I was effective when applied to either newly eclosed females prior to vitellogenic oöcyte development or to gravid females. Precocene I was also effective in decapitated females, indicating that the action of the compound is not mediated by the brain. Corpus allatum volume, presumably a reflection of secretory activity, increased between 0 and 24 hr after eclosion in control females but not in precocene-treated females even after 48 hr. However, when females were removed from precocene medium, gland volumes increased within 48 hr to approximately those of control flies. This result is consistent with the reversibility of the precocene effect on Drosophila adults. These results suggest that precocene acts on the corpus allatum of Drosophila adult females to produce juvenile hormone deficiency.     

Age-dependent physiological responses of last instarHelicoverpa zea larvae to oral and topical application of precocene II

Newly-ecdysed last instar larvae ofH. zea grouped into 100-, 200-, 300-, or 400-mg categories were fed diet containing precocene II or given precocene II topically on the abdomen. The time for larvae to reach a maximal weight, time to pupation, growth rate, and the amount of precocene II excreted were calculated. Younger larvae of lower weights, which were fed or topically treated with precocene II required more time to reach their maximal weight, had a lower maximal weight, a lower growth rate, and required more time to pupate than control larvae. Older larvae represented by the largest weight category were less sensitive to precocene II, had a shorter delay in reaching maximal weight, and a shorter delay in the time to pupation than control larvae; larvae in the largest weight category that were fed precocene II also had smaller decreases in the growth rate. Growth rate declines for larvae given topical doses of precocene II, however, were largest for the oldest larvae. All larvae given a single topical dose excreted precocene II for several days and were most efficient at eliminating smaller doses; larger, older larvae excreted more precocene II than smaller, younger larvae. Age-dependent responses to precocene II indicate that growth and metabolic processes, as well as xenobiotic metabolism, change in last instar larvae.     

Chemical allatectomy of late Locusta embryos by a synthetic precocene and its effect on hopper morphogenesis

The anti-allatin substance, 7-ethoxy-precocene II (= “precocene III”) was topically applied to eggs of Locusta migratoria migratorioides with fully grown embryos in stage XX (about 64 ± 4% of the whole period of egg development). A day after precocene application the eggs were washed for 10–15 s in acetone and then transferred to clean containers for removing precocene residues and for preventing contamination at hatching. The treatment induced prothetelic morphogenetic disturbances which became overt in the subsequent hoppers; the effect was dose dependent and the ED₅₀ (= effective anti-allatin dose 50%) was low, 20.5 μg precocene III per g fresh egg weight (= 0.37 μg per egg). Quite similar results were obtained following application of precocene III to eggs with embryos in stage XXI (73 ± 4% of the egg development). These findings and direct examination of histological sections of the embryonic corpora allata demonstrated that precocene chemically allatectomizes late Locusta embryos. The lethal effect of precocene III was dependent on the washing. When the eggs were washed in acetone a day after application, mortality did not occur in a dose-dependent way; even the highest dose applied, 256 μg precocene III per egg (= 14405 μg per g fresh weight), was less than the LD₅₀ (lethal dose 50%). In contrast, without washing mortality was dose dependent, but it occurred later, at or after hatching; the LD₅₀ was 1334.9 μg per g (= 22.7 μg/egg). The results show that the late embryos are highly susceptible to the anti-allatin effect of the precocene, but are extremely insusceptible to its lethal effect; toward hatching, however, susceptibility to the lethal effect becomes marked.With doses between 45–14405 μg precocene III per g fresh egg weight, the anti-allatin effect became overt by a quite-uniform belated morphogenetic response. All hoppers which hatched from precocenetreated eggs were morphogenetically normal in the 1st instar and in the beginning of the 2nd instar, but the duration of the 2nd instar was almost doubled and at the end of this instar over 96% of the locusts died in the moult, being unable to shed the exuvia. Artificial removal of the apolyzed old cuticle revealed 3rd instar prothetelic adultiforms. These results and some data in the literature indicate that allatectomy of the embryo does not result in prothetelic morphogenetic disturbances in the 1st and early 2nd instar larvae and may impose the question what is the role of the juvenile hormone in late embryos and early larvae.     

Precocene-induced degeneration of the corpus allatum of adult females of the bug Oncopeltus fasciatus

Application of precocene II, either topically or by contact method, to newly emerged adult females of O. fasciatus inhibits egg maturation and induces degeneration of the corpus allatum (CA). Seven days after precocene-treatment the CA were very small. Ultrastructural studies revealed a number of disintegrating nuclei, degenerating mitochondria, lysosome-like bodies and autophagic vacuoles. Fifteen days after precocene treatment the CA showed further reduction in the size and number of cells, and the degenerative changes were much more pronounced. It appears that the effect of prococene on the CA is irreversible.     

Morphogenetic effects of precocenes on three aphid species

The disruptive effects of precocenes I, II and III on metamorphosis were assessed in apterous viviparae of Acyrthosiphon pisum, Aphis fabae and Megoura viciae. Prenatally applied precocene I had no effect, precocene II caused supernumerary moulting (metathetely) in M. viciae but precocene III induced both precocious adult development (prothetely) and metathetely in all three species. the latter compound also promoted precocious adult development in alate virginoparae, males and ovipara-producers of M. viciae. However, gynoparae of A. fabae, and oviparae of M. viciae and A. pisum were resistant to this action. When topically applied to young adults of A. fabae or M. viciae none of the precocenes caused alate progeny to be produced but all did so in A. pisum. However, the previous supposition that precocene II promotes wing development because it decreases juvenile hormone titres should be viewed with caution as: (a) Precocene II was more potent than precocenes I and III when inducing wing formation but did not provoke precocious adult development, an overt indication of low juvenile hormone titres. (b) Precocene I and III were equally effective in inducing alate progeny but only the latter provoked precocious adult development. (c) Attempts to prevent or reduce the alata-inducing property of precocene II with juvenile hormone I were unsuccessful.     

Effect of precocene on development of ovarian follicles in flesh fly, Sarcophaga ruficornis F

Administration of precocene II (6,7-dimethoxy-2, 2-dimethyl chromene) to freshly emerged virgin female flies of S. ruficornis adversely affected the development and differentiation of ovarian follicles leading to a number of morphological abnormalities. Precocene treatment resulted into suppression of development of egg chamber, differentiation of follicular epithelium, degeneration of nurse cells, growth of oocyte and uptake of yolk granules by oocytes. The results suggest that precocene induced effects are due to deficiency of juvenile hormone.     

Hepatotoxicity of the anti-juvenile hormone precocene II and the generation of dihydrodiol metabolites

Precocene II (6,7-dimethoxy-2,2-dimethylchromene), the most active anti-juvenile hormone isolated from Ageratum houstonianum, has been shown to be hepatotoxic in male Sprague-Dawley rats. A single 300-mg/kg dose of precocene II administered via i.p. injection caused extensive necrosis of parenchymal cells in the hepatic centrolobular areas. Liver functions were markedly affected as shown by the significant increases of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase in the serum. By means of reversed-phase high pressure liquid chromatography (HPLC), [³H]precocene II was found to be rapidly metabolized in vitro by rat liver microsomes in an NADPH-generating system. Approximately 5% (3.4 nmol/mg protein) of the radioactivity from the [³H]precocene II substrate was covalently bound to the macromolecular pellet at the end of a 15-min incubation period when phenobarbital (PB)-induced microsomes were used. Results obtained from experiments using different incubation systems indicated the involvement of the cytochrome P-450-dependent monooxygenases in the metabolism of precocene II and the concurrent covalent binding. The most predominent metabolite was isolated and accounted for >90% of the radioactivity associated with the ethylacetate-extractable metabolites. Further analysis by mass spectrometry and proton nuclear magnetic resonance (NMR) spectroscopy identified it as a 37 : 63 stereoisomeric mixture of the cis and trans 3,4-dihydrodiols of precocene II. A highly reactive (3,4-epoxy-6,7-dimethyl-2,2-dimethylchromane (precocene-3,4-epoxide) was thus suggested as a crucial metabolic intermediate which may be responsible for the histopathological changes seen in rat liver.     

Precocene sensitivity of corpora allata in adult female Locusta migratoria after electrocoagulation of the pars intercerebralis neurosecretory cells

Destruction, by electrocoagulation, of the median neurosecretory cells of the pars intercerebralis of 2-day old adult female Locusta migratoria completely suppressed normal juvenile hormone-biosynthetic activity of the corpora allata in most animals. For example, 6 days after electrocoagulation the rates of spontaneous juvenile hormone biosynthesis, measured by radiochemical assay of freshly isolated glands, showed a median value of less than $\text{1}\text{100}$ that of the corresponding sham operated controls, which were then in mid-vitellogenesis. Injection of graded doses (200–1600 μg) of precocene I at this time, followed by assays five days later, resulted in a similar decline of both corpus allatum volume and precocene epoxidase activity (measured by radiochemical assays of precocene I dihydrodiol formation in vitro) in pars intercerebralis-coagulated and sham-operated animals, when expressed as a percentage of their own zero precocene controls. Electrocoagulation of the pars intercerebralis largely prevented the normal increase in both corpus allatum volume and its epoxidase activity, so that by age 13 days these parameters were about 2.5-fold lower in coagulated vs control (sham operated) animals. In fact, electrocoagulation had no marked effect on the value of epoxidase activity per unit corpus allatum volume. It is concluded that the corpora allata from this species and stage are sensitive to precocene irrespective of whether they are active in biosynthesis of juvenile hormone.     

Behavioral changes and growth inhibition in last instar larvae of Heliothis zea induced by oral and topical application of precocene II

In diet choice tests, newly-ecdysed fifth instar Heliothis zea larvae randomly chose diet containing 4 mM precocene II or diet without precocene II but initially preferred to feed on diet without precocene II. After two days, however, they were found more frequently on diet containing precocene II. Nevertheless, larval growth and development were inhibited when forced to eat diet containing precocene II. Removal of the maxillae, the site of the primary gustatory receptors, did not prevent the growth inhibition caused by larval consumption of diet with precocene II. Topical treatments with precocene II also caused larval growth inhibition, and daily treatments were more detrimental to the larvae than a large single dose. Addition of methylenedioxyprecocene (6,7-methylenedioxy-2,2-dimethylchromene) to the diet did not reverse the observed larval growth inhibition and this compound was itself significantly toxic. These results indicate that larval growth and development are disrupted after precocene II ingestion, but not because of preingestive discrimination by olfaction and gustation.     

Precocene II modifies maternal responsiveness in the burrower bug, Sehirus cinctus (Heteroptera)

Abstract. The anti-Juvenile Hormone agent precocene II was used to investigate the relationship of corpora allata activity to subsocial behaviour in a burrower bug Sehirus cinctus Palisot (Heteroptera: Cydnidae). Egg-brooding females treated with a range of dosages of precocene II exhibited reliably depressed maternal defensive behaviour when treated with at least 70 μg of precocene II, but attraction to eggs was only depressed at higher dosages. This effect was not due to precocene II toxicity, as demonstrated by the prevention of depression effects through simultaneous treatments of precocene II and the Juvenile Hormone analogue methoprene. Methoprene, however, failed to reinstate maternal responsiveness in maternally depressed females that had been previously treated with precocene II. This study provides the first clear evidence that insect parental behaviour can be modified by treatment with anti-Juvenile Hormone agents, and suggests that the role of the corpora allata in governing care in S. cinctus is different from that of other maternal insects, such as earwigs.     

Antigonadotropic and antiallatal effects of precocene II in the firebrat, Thermobia domestica (Thysanura,Lepismatidae)

The sensitivity of an apterygote insect to precocene II was investigated for the first time in females of Thermobia domestica. Topical applications of this compound, which has been said to possess anti-juvenile hormone activity, were carried out at various times in the postecdysial period of a reproductive cycle, during intense vitellogenesis in the terminal oöcytes. The effects of treatment were observed on mortality, fecundity, insemination and also on the ultrastructure of corpora allata; they depend on the dose used and on the day of treatment. A single application of 10 μg/insect at the beginning of the postecdysial period induces antigonadotropic effects in females reared both with and without males. The effects of precocene add to those of non-insemination, which also elicits inhibition of oöcyte maturation. In addition, the cytotoxic action of precocene, leading to degeneration of the corpora allata, which is most probably irreversible, was demonstrated.     

The role of exogenous JH I,JH III and Anti-JH (precocene II) on queen induction of 4.5-day-old worker honey bee larvae

Worker larvae at an age of 4½ days were fed one of several mixtures of reconstituted royal jelly adjusted to a refractive index of 1.3825 and supplemented with JH I, JH III or Anti-JH (precocene II). In addition, juvenile hormone was topically applied to larvae of the same age. It was readily apparent that caste induction is concentration-dependent and that 4?-day-old worker larvae can still develop into queens under laboratory conditions, providing that they have not stopped feeding or can be induced to commence feeding again. These findings are contrary to the general belief that queen induction is not possible after a socalled sensitive period of 3–3½ days. Queens resulted only from honey bee larvae exposed to royal jelly containing 1 μg of JH I. In addition, oral application at this concentration resulted in the only case in which the normal mean weights of worker honey bees were exceeded. All other concentrations of juvenile hormone were not sufficient to initiate queen induction, although its lower concentration may have influenced the production of intercastes.Precocene II did not play a role in queen induction and it also did not interfere with the growth of developing larvae or adults. In addition, the lack of malformations in honey bees treated with precocene II indicates that the use of such a compound as a control agent in insect populations will probably not be detrimental to honey bee larvae that are at least 4½ days old. However, large doses of precocene will quickly kill most 3½-day-old honey bee larvae.The evidence presented here clearly indicates that caste determination is regulated by the endocrine system in honey bee larvae. Food intake in honey bee larvae may well be regulated by the endocrine system. Thus, an apparently inhibited corpus allatum (C.A.) could be reactivated by food intake coupled with juvenile hormone. The food intake restriction that worker larvae normally encounter in the hive probably results in a cessation of C.A. activity. The increase in food intake by queen larvae, on the other hand, carries an increase in growth and accompanying morphological changes necessary for queen development. This concept may also explain the development of intercastes encountered in in vitro studies. Only those larvae that follow a normal food intake sequence, i.e. moderate during the first 3–4 days or so, will develop into queens. Conversely, those larvae that take in too much food during the early portion of development may achieve incomplete development of the neurosecretory system and, thus, develop into intercastes.     

Precocene II-induced alate production in isolated and crowded alate and apterous virginoparae of the aphid, Macrosiphum euphorbiae

Precocene II was applied at doses ranging from 0.05 to 0.70 μg per individual to newly moulted adult alate and apterous virginoparae of Macrosiphum euphorbiae kept isolated or in groups of 10 per plant, under an 18L:6D photoperiodic regime. While isolated controls of both morphs produced exclusively apterous progeny, alatae virginoparae were produced in generally dose-dependent proportions by precocene-treated individuals. Grouped controls of both morphs produced alatiform progeny as expected, but in precocene—treated groups, the proportions of alate progeny generally increased as a function of dose. The overall proportions of alate offspring produced, and numbers of days after treatment when morph production was affected, were generally greater for alatae than for apterae, indicating a greater sensitivity to precocene in alatae. However during the first few days after treatment, the alatizing effect of precocene was stronger for apterae, suggesting that the first embryos produced by alatae were irreversibly determined as apterae.In an experiment where isolated alatae and apterae received 0.5 μg of precocene II at different ages ranging from 1 to 13 days after the adult moult, the alatizing effect of the compound, measured by the persistence of alate production, varied with age and morph. While in alatae, the persistence decreased more or less regularly with age, in apterae it initially increased to a maximum in the middle of reproductive life, and subsequently decreased. The results provide support for the hypothesis that juvenile hormone is involved in regulating alary dimorphism in M. euphorbiae.     

The external morphology and distribution of cuticular hair organs on the claws of the American lobster, Homarus Americanus (Milne-Edwards)

Four classes of microscopic cuticular hair organs were found on the chelae of Homarus americanus (Milne-Edwards). Type I and Type II organs possess long single sensillae, 30–60 μm and 70–130 μm, respectively. Type III organs are toroid bumps 20–30 μm in diameter, with a small tuft of fibers projecting from the center. Type IV organs are small conical hairs ≈ 1 μm in length.Type IV organs were uniformly distributed over the claws. The distributions of Types I, II, and III organs were analysed through multivariate analysis of variance (MANOVA) with respect to claw, side, and area. Type I organs showed significance of the three-way interaction only. Type II organs showed significance of side, area, and the claw by area interaction. Type III organs showed significance of area, and all interactions. Changes in number and density of Types I, II and III organs, and in number of Type IV organs, were found with growth.     

Biomorphogenetic changes caused by the application of precocene II in nymphs of Triatoma infestans (Klug, 1834) (Hemiptera, Reduviidae, Triatominae)

The morphogenetic effect of precocene II on 4th instar nymphs of Triatoma infestans was examined. Topical treatment with precocene II (200, 300 and 400 micrograms/nymph) induced the formation of adultoids with the following characteristics: rudimentary wings, three segmented tarsi, ocelli, deformed genitalia and mouth-parts and testes with intermediary development. Precocene II also increases the duration of the molting cycle. The survival of the adultoid insects was related to the inverse of the applied doses. These data extend our knowledge of the action of precocene II on triatominae species.     

Comparative studies on pharmacokinetics and metabolism of the anti-juvenile hormone precocene II

The cuticular penetration and pharmacokinetics of the anti-juvenile hormone precocene II were determined in a sensitive species (Oncopeltus fasciatus) and an insensitive species (Heliothis zea). Precocene was sequestered by the fat body and slowly metabolized in Oncopeltus, but rapidly metabolized and excreted in Heliothis. Studies in vitro using inhibitors for cytochrome P-450 and for cyt P-450-NADPH-reductase, confirmed the anticipated detoxification of precocene by a mixed-function oxidase via the 3,4-epoxide. Use of the inhibitors in vivo had no influence on the metabolism of precocene.     

Effect of precocene II on fatty acid metabolism in the pea aphid, Acyrthosiphon pisum, under cold stress

Pea aphids, Acyrthosiphon pisum (Harris), reared at 10 degrees C contain higher levels of fatty acids than those reared at 25 degrees C. This is primarily the result of an accumulation of triacylglycerols containing myristic acid. When aphids reared at 25 degrees C were transferred to 10 degrees C there was a gradual increase in triacylglycerol content that reached a maximum at 16 days post-transfer. Treatment of aphids with precocene II prior to transfer to 10 degrees C blocked the accumulation of fatty acids including myristic acid. A single application of 2 microg precocene II/aphid or two applications of 0.5 microg precocene II/ aphid administered on consecutive days resulted in aphids moved to 10 degrees C maintaining the same fatty acid profile as aphids maintained at 25 degrees C. Aphids that were treated with precocene II and maintained at 25 degrees C did not show changes in fatty acid profiles. Rearing aphids at 10 degrees C resulted in lower rates of reproduction and lower total numbers of progeny with longer longevity. Treatment with precocene II significantly decreased the total number of progeny produced at both temperatures. Precocene II did not reduce life span of aphids reared at 25 degrees C, however, the life span of treated aphids reared at 10 degrees C was decreased. The mechanism by which precocene II prevents the accumulation of myristic acid in aphids reared at 10 degrees C remains to be determined.     

Antijuvenile influence of the precocene on the development of adult antennae in the large fruit-tree tortrix Archips podana Scop. (Lepidoptera: Tortricidae)

The influence of precocene II, an antijuvenile agent, on the development of adult antennae in the large fruit-tree tortrix A. podana Scop. was demonstrated. Treatment of the fifth instar larvae and prepupae with different doses of precocene proved to cause different sensitivity of the specimens to the juvenile hormone deficit. Treatment with 450 and 600 ??g precocene per specimen during the first days after ecdysis to the fifth instar caused the death of larvae. Treatment with 300, 450, and 600 ??g per specimen on the third day of the fifth instar larvae and prepupae caused a delay in the development of adult antennae. The results are discussed with respect to the role of the juvenile hormone in the development of imaginal structures during metamorphosis.