Effect of Azadirachtin on vitellogenesis of Labidura riparia (Insect Dermaptera)

This study investigates the effects of the insect growth regulator Azadirachtin (AZA) on the ultrastructure of ovaries and fat body of the earwig Labidura riparia. Ovarian development is severely reduced in AZA-injected females in a dose-dependent manner. Follicles exhibit degenerative changes, separation of follicle cells from the oocyte, and lack of pinocytotic vesicles as of yolk spheres in cortical ooplasm. Adipocytes show fragmented rough endoplasmic reticulum (RER), numerous autophagic vacuoles, multivesicular bodies, osmiophilic lipid droplets, and many large glycogen areas. Gel electrophoresis reveals that vitellogenin is absent from both fat body and hemolymph, and that vitellin is not deposited in the ovary. These pathological effects are not linked to an absence of feeding. The effect of AZA on vitellogenesis is rescuable by Juvenile hormone (JH) treatment. The inhibition of vitellogenesis by AZA is discussed on the basis of its direct cytotoxic effect as well as its interference with the neuroendocrine system.     

Ultrastructural changes in the corpus allatum after azadirachtin and 20-hydroxyecdysone treatment in adult females of Labidura riparia (Dermaptera)

In previous reports, we have shown that the injection of azadirachtin (AZA) as well as 20-hydroxyecdysone (20E) into vitellogenic females of Labidura riparia induces inhibition of vitellogenin synthesis and ovarian development. Juvenile hormone (JH) treatment rescues vitellogenin synthesis and ovarian growth (Sayah et al., 1995, 1996). In this work, we have studied ultrastructural changes of corpus allatum (CA) after injection of 200, 400, and 600 ng of 20E or 1, 3, and 5 microg of AZA. CA cells exhibit signs of inactivity in both AZA and females treated with 20E at doses of 3 microg and 400 ng, respectively. Conspicuous cytological effects consisting of multivesicular bodies with dense contents, abnormally large intercellular spaces comprising myelinic structures, and rare smooth endoplasmic reticula occurred in cytoplasm of CA glandular cells in both experimental females. However, the CA ultrastructure of females injected with 20E differs from CA cells of females injected with AZA in having a cytoplasm containing numerous electron-lucent intracellular areas and marked glycogen zones. They also differ in having abundant microtubules and well-developed junctional membranes. At a dose of 600 ng of 20E or 5 microg of AZA, the intensity of the cytotoxic effects is more apparent. CA cells display pycnotic nuclei, spherical mitochondria, large multivesicular bodies, and vacuolization of the cytoplasm. These results are discussed and compared with observations made on other insect species.     

Artifactual stimulation of vitellogenesis in Aedes aegypti by 20-hydroxyecdysone

Single or repeated, non-physiological, high doses (0.5–5.0 μg/female) of 20-hydroxyecdysone or ecdysone injected into sugar-fed female Aedes aegypti stimulated follicular growth and deposition of yolk, but suppressed accumulation of protein yolk to approximately one-third, and lipid yolk to one-half that in an equal number of follicles with equivalent yolk length taken from blood-fed controls. Physiological doses (500 pg/female) of ecdysone or 20-hydroxyecdysone or the implantation of ecdysone-secreting ovaries (verified by bioassay), into sugar-fed females failed to induce any yolk deposition. In these experiments, yolk precursors were not the limiting factor, because in decapitated females, digesting a blood meal, the injection of a physiological dose of 20-hydroxyecdysone or the implantation of ecdysone-secreting ovaries still did not stimulate vitellogenesis. Finally, continuous infusion of 500 pg or even 50 ng 20-hydroxyecdysone/hr for 22 hr was as ineffective as single or multiple injections of equivalent doses of hormone. Consequently, rapid excretion or catabolism of 20-hydroxyecdysone by the sugar-fed female does not explain the need for high doses to induce vitellogenesis, or the failure of oöcytes to mature with normal protein and lipid content. Apparently, ovarian ecdysone is not the factor by which normal vitellogenesis is initiated and maintained in this mosquito.     

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick,Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.     

Hormones regulating structural changes in the adipocytes of the female earwig Labidura riparia

Changes in periovarian fat body fine structure occur during the reproductive cycle of the female earwig Labidura riparia. During the process of vitellogenesis, high levels of protein synthesis are seen in the adipocytes. Simultaneously, the formerly osmiophilic lipid droplets become non-osmiophilic, while an osmiophilic material diffuses from lipid droplets to the cisternae of the rough endoplasmic reticulum (RER). These structural features alter during non-vitellogenic periods: RER and Golgi apparatus regress and seem to be inactive. Lipid droplets are once again osmiophilic and no material enters into RER cisternae. Using microsurgical manipulations, hormonal treatments and light and electron microscopy, we have investigated the regulation of these changes. The neuroendocrine cerebral centre pars intercerebralis, by the action of the juvenile hormone of the corpus allatum. triggers protein metabolism. Another neuroendocrine center, the pars lateralis. and the hormone 20-hydroxyecdysone, regulate structural lipid droplet changes in the adipocytes.     

Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

Female Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20-hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood-fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole-body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production. Implantation of ecdysone-secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone-secreting ovaries resulted in normal egg development. These experiments indicate that the appearance of JH precedes 20-hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.     

Filariomyces forficulae: occurrence and effects on the predatory earwig, Labidura riparia

Filariomyces forficulae occurs commonly on the earwig, Labidura riparia, in Florida. The hypopial stage of a mite, Histiostoma sp., often associated with L. riparia is also sometimes infected by the same fungus. Some other earwig species that occur with L. riparia were not found to be hosts. Sites of fungal infection on the earwig were highly variable and influenced by several factors. F. forficulae was transmitted by several types of insect contact including mating with varying efficiencies and by a mechanical inoculation technique. Developmental time required by the fungus from ascospore to mature perithecia was 10–12 days at 24°C. In laboratory studies there were small but consistent differences between longevities of infected and noninfected groups of earwigs and infected populations of earwigs died sooner than noninfected ones. Infection resulted in differences in pattern of egg production; however, no numerically significant differences in total egg production or egg-hatching ability between the two groups were found.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

Identification of 10 microsatellite loci in the earwig Labidura riparia (Dermaptera,Labiduridae)

Ten microsatellite loci were isolated from the earwig Labidura riparia (Pallas, 1773). The polymorphism of the loci was assessed in 24 individuals from one population. The number of alleles ranged from four to 11 alleles and observed and expected heterozygosities from 0.250 to 0.833 and from 0.551 to 0.861, respectively.     

Dopamine and octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). We have also measured the levels of 20E in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). We have studied the effects of JH application on 20E level in 2-day-old wt females of D. virilis and demonstrated that an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed.     

Induction of choriogenesis by 20-hydroxyecdysone in the German cockroach

Experiments in vitro have shown that 20-hydroxyecdysone (at a concentration of 0.2 and 2 muM and after 12 and 24 hr of incubation) is able to induce the precocious deposition of chorion materials by the follicular epithelium of young oocytes of the cockroach Blattella germanica. Since previous studies had shown that 20-hydroxyecdysone levels in B. germanica ovaries increase with oocyte maturation to reach a peak just before oviposition, we therefore hypothesize that ovarian ecdysteroids trigger Choriogenesis in this species through an autocrine action.     

Activity of corpora allata,endocrine balance and reproduction in female Labidura riparia (Dermaptera)

Summary The reproductive activity of Labidura riparia females involves, after a 5-day maturation stage, a regular alternation of ovarian cycles and egg-care stages averaging 10 days each. Vitellogenesis is characterized by an increase in the size of the corpora allata (CA) where structured SER bodies appear, and by a rise of juvenile hormone (JH III) content in the hemolymph which is followed by an increase in the level of ecdysteroids. During the egg-care periods, the CA are inactive; structured bodies generate autophagic vacuoles, the titer of JHs and later that of ecdysteroids in the hemolymph decreases and remains stationary. Ovariectomy causes hypertrophy and hyperactivity of the CA for about two months. Subsequently, the titer of JH decreases and old females may display parental behaviour; the level of ecdysteroids falls and remains unchanged. After cauterization of the pars intercerebralis (PI) of the protocerebrum, the ovarian activity stops, the ovary shrinks, the JHs rapidly disappear but ecdysteroids remain at the same or even higher levels than those of normal females of the same age. On the basis of these data, we postulate the existence of a center located in the PI, inhibiting the production of ecdysteroids, and of a stimulating center located outside this area. The PI also exhibits an allatotropic function.

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Résumé L'activité sexuelle des femelles de Labidura riparia comporte au terme d'une phase de maturation sexuelle de 5 jours une alternance régulière de cycles ovariens et de phases de soins aux oeufs d'une durée moyenne de 10 jours. La vitellogenèse est caractérisée par l'accroissement des CA oú apparaissent des amas structurés de RE, par une élévation du titre hémolymphatique de la JH III, puis des ecdystéroïdes. En période de soins aux oeufs, les CA sont inactifs (rejet des amas de reticulum sous la forme de vacuoles autophagiques), le titre hémolymphatique de l'hormone juvénile puis des ecdystéroïdes diminue puis reste stationnaire. L'ovariectomie détermine une hypertrophie et pendant 2 mois environ une hyperactivité des CA; ultérieurement, le titre de l'hormone juvénile diminue et les femelles âgées peuvent manifester un comportement parental; le titre des ecdystéroïdes ne s'annule pas chez les femelles ovariectomisées. Après parsectomie, l'activité ovarienne cesse, l'ovaire régresse, le titre de l'hormone juvénile s'annule rapidement mais les ecdystéroïdes restent présents à des titres égaux, voire supérieurs à ceux appréciés chez les femelles normales de même âge. Ces données permettent d'envisager l'existence d'un centre inhibiteur de la production des ecdystéroïdes situé dans la PI et d'un centre excitateur situé en dehors de cette région. La PI a également une fonction allatotrope.

     

Allatostatin-like peptide in the brain-retrocerebral complex of the earwig Labidura riparia: cyclic variations related to the reproductive cycle

Summary

A polyclonal antibody raised against allatostatin-3 of Blattella germanica (BLAST-3) has been used to immunolocalize allatostatin-like peptides in the brain-retrocerebral complex of Labidura riparia adult females. Strongly stained immunoreactive cells are observed in the pars intercerebralis (14 cells) and mainly in the pars lateralis (32 cells). Fibres leading to the corpus allatum are also stained. In the deutocerebrum, one cell is immunostained at the root of each antennal nerve. In the tritocerebmm two cells in each brain hemisphere are weakly immunostained. During the reproductive cycle, these cells and their axons show immunoreactivity at previtellogenic, ovulation and ovarian arrest periods. During vitellogenesis, immunoreactivity is restricted to only four perikarya in the pars intercerebralis.

When young vitellogenic females are injected with 20-hydroxyecdysone (20E), which inhibits vitellogenesis, full immunoreactivity reappears, suggesting sensibility of these cells to 20E as is expected for a negative feed-back loop (Sayah et al., 1995).

These results show that BLAST-3-like material is produced periodically in Labidura in correlation with low levels of juvenile hormone and the absence of vitellogenesis. This study contributes to provide information on the degree of homology of allatostatins across various insects.     

Changes in the lipid and fatty acid composition of hemolymph and ovaries during the reproductive cycle of Labidura riparia

Lipid metabolism was investigated during the reproductive cycle of Labidura riparia (Pallas). The lipid classes and their constitutive fatty acids present in hemolymph and ovaries were measured using thin‐layer chromatography and gas‐liquid chromatography. In the hemolymph, total lipids increase steadily from the previtellogenic period to vitellogenic arrest. These lipids are predominantly diacylglycerols and phospholipids. In the ovaries, total lipids increase during vitellogenesis then decrease during the vitellogenesis arrest period. The major lipids are triacylglycerols, followed by phospholipids. In both hemolymph and ovaries, all lipid classes contained variable proportions of seven main fatty acids: the saturated fatty acids myristic acid (14:0), palmetic acid (16:0), and stearic acid (18:0); the monounsaturated fatty acids palmitoleic acid (16:1) and oleic acid (18:1); and the polyunsaturated fatty acids linoleic acid (18:2) and linolenic acid (18:3). Unsaturated fatty acids predominate throughout the reproductive cycle. The percentage compositions of total and triacylglycerol fatty acids do not change markedly during the reproductive cycle in hemolymph nor in ovaries, with 18:2, 18:1 and 16:0 fatty acids being the major components. However, for diacylglycerols and phospholipids, the proportions of fatty acids vary systematically. For phospholipids during the vitellogenesis period, 18:2 increases considerably whereas other fatty acids decrease; for diacylglycerols, these fatty acids vary in the reverse way.     

In vivo and in vitro effects of 20-hydroxyecdysone and methoprene on diapause maintenance and reproductive development in Musca autumnalis

Abstract. Face flies overwinter as adults in reproductive diapause. Administration of 20-hyroxyecdysone and/or methoprene induced reproductive development in diapausing flies which were maintained in a diapause-inducing environment. Hormone effects were additive and female flies were more sensitive than males. Release of vitellogenin from cultured fat body was stimulated by 20-hydroxyecdysone or methoprene. Transfer of flies from diapause to diapause-breaking environments induced some to break diapause, but this decreased with the time flies had been in a diapause-inducing environment. In contrast, topical application of methoprene to diapausing flies induced reproductive development irrespective of their ages even when they were kept in the diapause-inducing environment for 80 day degrees above a 12°C base temperature (14.5 days). Therefore diapause induction must depend on hormone levels less than some threshold level. The putative threshold varied according to diapause propensities of different genetic lines. Lines showing high frequencies of diapause required greater amounts of methoprene for reproductive development in diapause conditions than did lines showing low frequencies of diapause.     

In vivo inhibition of aldehyde dehydrogenase by disulfiram

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein–drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of ∼71 and ∼100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH⁺=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃, derived from the enzyme active site region, and were modified at Cys₃₀₂ by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).     

In vivo inhibition of aldehyde dehydrogenase by disulfiram

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+)=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).     

In vivo inhibition of aspartate aminotransferase in mice by L-hydrazinosuccinate

L-Hydrazinosuccinate, which has been shown to be a slow-, tight-binding inhibitor of aspartate aminotransferase (EC 2.6.1.1) in vitro, was tested as an inhibitor in vivo of the enzyme as well as other pyridoxal enzymes. Intraperitoneal administration to mice at a dose of 0.6 mmol/kg rapidly decreased aspartate aminotransferase activities in liver and kidney cytosols to a minimal level lower than 10% of the original, and no appreciable reversal of the inhibition was observed after 24 h; at lower doses the activities were significantly recovered during the same period following an initial marked decrease. Of the other pyridoxal enzymes tested, alanine aminotransferase in liver was the most sensitive to the inhibitor. It was initially inhibited as severely as aspartate aminotransferase, but the inhibition was reversed considerably faster. Aspartate aminotransferase activities in brain and heart were less severely affected than those in liver and kidney; they were less markedly lowered initially and were substantially recovered after 24 h. Consistent with the observed organ specificity, heated extracts from brain and heart in the mice administered with the inhibitor showed relatively weak inhibitory activities in vitro to aspartate aminotransferase purified from pig heart, while the extracts from liver and kidney were strongly inhibitory.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone during pupal—adult metamorphosis of Mamestra configurata wlk

Large peaks of ecdysone (E, 2,875 ng/g live wt) and 20-hydroxyecdysone (20-HE, 2,150 ng/g live wt) occur on days 8 and 12, respectively, of postdiapause pupal-adult metamorphosis at 20°C in the bertha armyworm, Mamestra configurata, and then decline to low levels (< 100 ng/g live wt) prior to eclosion of the moth (50% eclosion at day 31.8). These peaks of E and 20-HE can be suppressed by treating the developing pupae with a physiological dose (2,500 ng/g live wt) of 20-HE. Suppression of E and 20-HE by 20-HE treatment was dose dependent, rapid (within 24 h of treatment) and permanent. The peaks of E and 20-HE were suppressed by 20-HE treatment on days 1, 3, 5, and 7 but the 20-HE peak was not suppressed by treatment on days 9 or 11. It is proposed that the mechanism by which 20-HE suppresses the production of E and thereby its own production forms a negative feedback loop that operates during the first 0.4 units of pupal-adult development in M. configurata. The function of the transitory peaks of E and 20-HE that form this feedback loop is currently unknown. Since most adults from pupae that had their ecdysteroid levels experimentally suppressed by 20-HE treatment were morphologically normal, it seems that the peaks of E and 20-HE have little or no function in controlling morphological development in pupae of M. configurata.