Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

Patterns of biosynthesis and accumulation of hydrocarbons and contact sex pheromone in the female german cockroach,Blattella germanica

De novo synthesis of contact female sex pheromone and hydrocarbons in Blattella germanica was examined using short in vivo incubations. Accumulation of pheromone on the epicuticular surface and the internal pheromone titer were related to age-specific changes in hydrocarbon synthesis and accumulation in normal and allatectomized females. The incorporation of radiolabel from [1-¹⁴C]propionate into the cuticular methyl ketone pheromone fraction was positively related to corpora allata activity during two gonotrophic cycles. During peak pheromone production the total internal lipid fraction contained greater titers of pheromone than the cuticular surface, and it too exhibited a cycle internally, preceding the rise in external pheromone. This suggests that synthesis and accumulation of pheromone internally are followed by transport of pheromone to the epicuticular surface where it accumulates. Radiolabel was incorporated efficiently into both cuticular and internal hydrocarbons after the imaginal molt and until the peak of pheromone synthesis, but it declined to lower levels before ovulation and throughout pregnancy. The internal hydrocarbon titer decreased 58% after oviposition, suggesting deposition in the egg case. It remained relatively unchanged during pregnancy and increased again during the second gonotrophic cycle. In allatectomized females, hydrocarbon synthesis was reduced relative to control females until oviposition in the latter. However, subsequent rates of hydrocarbon synthesis in allatectomized females (without oothecae) exceeded the rates in sham-operated females (with oothecae). In the absence of ovarian uptake of hydrocarbons, the internal titer increased without the decline found in control females at oviposition. As internal hydrocarbons increased, so did cuticular hydrocarbons and both internal and cuticular methyl ketone pheromones. These patterns corresponded well with feeding patterns in sham-operated and allatectomized females, suggesting that pheromone production is normally regulated by stage-specific feeding-induced hydrocarbon synthesis (precursor accumulation internally) and juvenile hormoneinduced conversion of hydrocarbon to pheromone. They also suggest that both the cuticle and the ovaries might be target sites for hydrocarbon and possibly methyl ketone deposition. © 1994 Wiley-Liss, Inc.     

Endocrine regulation of female contact sex pheromone production in the German cockroach, Blattella germanica

ABSTRACT The amount of the major component of the cuticular sex pheromone, 3, 11-dimethyl-2-nonacosanone, on individual female German cockroaches, Blattella germanica (L.), as a function of age was determined by gas-liquid-chromatographic analysis. Accumulation of phermone increased with age in both virgin and mated females. During the first gono-trophic cycle, the pheromone accumulated most rapidly when oocyte growth rates were maximal (days 5–10), and least rapidly while the female carried an ootheca (days 11–32). Pheromone accumulation was similar in virgin and mated females when the same physiological stages (determined by oocyte maturation) were considered. Inhibition of Juvenile Hormone release, through allatectomy, chemicals (precocene or fluoromevalonate), or through mechanical egg case implants, suppressed or delayed pheromone production and oocyte growth. The Juvenile Hormone analogue ZR512 induced allatectomized or head-ligated females and females with chemically or mechanically inhibited corpora allata to produce pheromone and enlarge their basal oocytes. Finally, ZR512 applied to intact females stimulated pheromone production in a dose-dependent manner.     

Time-course haemolymph juvenile hormone titres in solitarious and gregarious adults of Schistocerca gregaria, and their relation to pheromone emission, CA volumetric changes and oocyte growth

The haemolymph JH III titres in solitarious and gregarious adult desert locusts, Schistocerca gregaria, were examined in relation to corpus allatum (CA) volumes, aggregation-maturation pheromone production in males and oocyte growth in females. The JH titres of gregarious females were generally higher than those of solitarious females at all ages studied. The titre patterns, however, were similar: relatively high on day 10, dropping to low levels between days 20 and 25, before rising again by day 25. In the solitarious males, the JH titre was very low on day 10 after fledging, but increased gradually and reached a maximal amount on day 30. The JH titre in gregarious males was low on day 10, elevated on day 15 coinciding with the start of the production of the pheromone, and dropped to a relatively low level on day 20 around the time of maximal pheromone production, then rising again by day 25. These results suggest that biosynthesis of the pheromone is associated with a high JH titre peak in the haemolymph. Although a clear relationship was found during the first gonadotropic cycle between JH titres, on one hand, and CA volume and oocyte growth, on the other, in both phases, no such correlation could be discerned in the second cycle.     

Juvenile hormone induction of pheromone gland PBAN-responsiveness in Helicoverpa armigera females

The maturation of corpora allata (CA) and the competence of pheromone glands in the adult moth Helicoverpa armigera, are both age-related and appear to be correlated. Sex pheromone glands of pharate adults do not produce sex pheromone independently, nor do they respond to exogenous PBAN. Newly emerged moths produce significantly less pheromone than day one moths. JH (juvenile hormone) II was found to be the main JH form produced by CA in vitro. JH II primed pheromone glands of pharate adults to respond to PBAN. In addition, injection or topical application of JH II to newly-emerged females induced pheromone production in the presence of PBAN. Our findings suggest that JH is involved in the initiation of pheromone production of Helicoverpa armigera.     

In vitro biosynthesis of JH III by the corpora allata of adult females of Blattella germanica (L)

A radiochemical assay which fulfills the required validation criteria has been used for quantification of the in vitro biosynthesis of JH III by the corpora allata of adult females of Blattella germanica throughout the 7 days of the first reproductive cycle. The presence of JH III has been independently confirmed by HPLC and mass spectrometry. Results indicate that rates of JH release increase repidly from day 3 to day 6, which is correlated with oocyte growth. The highest levels of JH release (2.58 ± 1.11 pmol/hr per pair) were obtained from day-6 females. The time course of JH production by CA from day-6 females showed that CA released JH at a linear rate for at least 9 hr. From these results, it can be concluded that titers at high production ages and linearity ranges are satisfactory enough to be used in studies on the regulation of JH production in this species.     

Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: The search for a unifying arthropod endocrinology

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.     

Juvenile hormone biosynthesis,oocyte growth and vitellogenin accumulation in Choristoneura fumiferana and C. rosaceana: a comparative study

We assessed the effects of age and mating status on in vitro juvenile hormone (JH) biosynthesis, oocyte growth, egg production and vitellogenin (Vg) accumulation in the tortricid moths, Choristoneura fumiferana and C. rosaceana. To determine whether vitellogenesis is dependent on the presence of JH, we also examined the effects of decapitation and JH analog treatments on egg production. In both species, the corpora allata (CA) of adult females released fmol quantities of JH, with JH II being the major homolog produced. The CA began producing detectable quantities of JH around the time of emergence. Full activation of the CA was observed a few hours sooner in C. fumiferana than in C. rosaceana. In pharate adults and young virgin females of both species, growth of the basal oocyte reflected changes in CA activity. Decapitation of newly emerged females significantly reduced egg production, but treatment of decapitated females with the JH analog methoprene resulted in egg production that was similar to (C. fumiferana) or greater than (C. rosaceana) that of controls, indicating that JH is required for oocyte maturation. Vg was first observed in the hemolymph before the presumptive time of CA activation, suggesting that the synthesis of this protein is not dependent on JH. The presence of normal quantities of Vg in the hemolymph of pupae decapitated before CA activation confirmed this hypothesis. The Vg titer underwent a transient decline following CA activation and was significantly lower in mated than in virgin females of both species 3 and 5 days after copulation. Since CA activation at emergence and mating are both expected to cause a rise in the JH titer, we suggest that the declines in the levels of Vg result from JH-enhanced Vg uptake by the developing oocytes. Mating induced a significant increase in egg production but had no measurable impact on rates of JH biosynthesis in vitro.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

Effects of chilling stress on allatal growth and juvenile hormone synthesis in the cockroach, Diploptera punctata

During the ovarian cycle of the cockroach, Diploptera punctata, a mitotic wave occurs in the corpora allata before an increase in gland volume and juvenile hormone (JH) synthesis. Previous studies have demonstrated that the brain inhibits mitosis and JH synthesis in corpus allatum (CA) cells until adult females have mated. Herein, we report that chilling stress effectively suppresses mating induced proliferation of CA cells. In mated females, chilling on melting ice for 0.5-3 hours caused a strong, dose-dependent decrease in mitotic activity. In insects chilled for 3 hours, although the mitotic wave in the CA was practically abolished, CA volume and JH synthesis finally reached peak levels typical of unchilled insects, despite a 2-day delay. Consequently, oocyte maturation and oviposition were also delayed by 2 days, yet in both chilled and unchilled insects, peak values of basal oocyte length were the same. By allowing virgin females to mate on different days after chilling, we found that the chilling effect could be retained in the insect body for at least 2 days. During this period, signals from mating could not effectively remove inhibition of CA cell proliferation. Unilaterally disconnecting the CA from the brain revealed that chilling stress mediated CA cell proliferation via the brain, and did not directly affect the CA.     

Stimulatory activity of cysteamine on juvenile hormone release in adult females of the cockroach, Blattella germanica

1. A study of the activity of cysteamine in relation to juvenile hormone (JH) production in adult females of Blattella germanica was carried out. 2. In vivo assays showed that cysteamine stimulates protein synthesis in the left colleterial gland and, in some instances, enhances oocyte growth. 3. In vitro assays demonstrated that cysteamine enhances JH release by incubated corpora allata (CA), and that this effect is more pronounced when using CA from 10-day-old females (period of ootheca transport), either connected to the corpora cardiaca (CC) or to the CC and to the brain. 4. Possible antiallatostatic effects of cysteamine are discussed.     

Identification of Differentially Expressed Genes in the Pheromone Glands of Mated and Virgin Bombyx mori by Digital Gene Expression Profiling

Background

Mating decreases female receptivity and terminates sex pheromone production in moths. Although significant progress has been made in elucidating the mating-regulated inactivation of pheromone biosynthesis-activating neuropeptide (PBAN) secretion, little is known about the mating induced gene expression profiles in pheromone glands (PGs). In this study, the associated genes involved in Bombyx mori mating were identified through digital gene expression (DGE) profiling and subsequent RNA interference (RNAi) to elucidate the molecular mechanisms underlying the mating-regulated gene expression in PGs.

Results

Eight DGE libraries were constructed from the PGs of mated and virgin females: 1 h mating (M1)/virgin (V1) PGs, 3 h mating (M3)/virgin (V3) PGs, 24 h mating (M24)/virgin (V24) PGs and 48 h mating (M48)/virgin (V48) PGs (M48 and V48). These libraries were used to investigate the gene expression profiles affected by mating. DGE profiling revealed a series of genes showing differential expression in each set of mated and virgin female samples, including immune-associated genes, sex pheromone synthesis-associated genes, juvenile hormone (JH) signal-associated genes, etc. Most interestingly, JH signal was found to be activated by mating. Application of the JH mimics, methoprene to the newly-emerged virgin females leaded to the significant reduction of sex pheromone production. RNAi-mediated knockdown of putative JH receptor gene, Methoprene tolerant 1 (Met1), in female pupa resulted in a significant decrease in sex pheromone production in mature females, suggesting the importance of JH in sex pheromone synthesis.

Conclusion

A series of differentially expressed genes in PGs in response to mating was identified. This study improves our understanding of the role of JH signaling on the mating-elicited termination of sex pheromone production.     

Cuelure but not zingerone make the sex pheromone of male Bactrocera tryoni (Tephritidae: Diptera) more attractive to females

In tephritid fruit flies of the genus Bactrocera Macquart, a group of plant derived compounds (sensu amplo ‘male lures’) enhance the mating success of males that have consumed them. For flies responding to the male lure methyl eugenol, this is due to the accumulation of chemicals derived from the male lure in the male rectal gland (site of pheromone synthesis) and the subsequent release of an attractive pheromone. Cuelure, raspberry ketone and zingerone are a second, related group of male lures to which many Bactrocera species respond. Raspberry ketone and cuelure are both known to accumulate in the rectal gland of males as raspberry ketone, but it is not known if the emitted male pheromone is subsequently altered in complexity or is more attractive to females. Using Bactrocera tryoni as our test insect, and cuelure and zingerone as our test chemicals, we assess: (i) lure accumulation in the rectal gland; (ii) if the lures are released exclusively in association with the male pheromone; and (iii) if the pheromone of lure-fed males is more attractive to females than the pheromone of lure-unfed males. As previously documented, we found cuelure was stored in its hydroxyl form of raspberry ketone, while zingerone was stored largely in an unaltered state. Small but consistent amounts of raspberry ketone and β-(4-hydroxy-3-methoxyphenyl)-propionic acid were also detected in zingerone-fed flies. Males released the ingested lures or their analogues, along with endogenous pheromone chemicals, only during the dusk courtship period. More females responded to squashed rectal glands extracted from flies fed on cuelure than to glands from control flies, while more females responded to the pheromone of calling cuelure-fed males than to control males. The response to zingerone treatments in both cases was not different from the control. The results show that male B. tryoni release ingested lures as part of their pheromone blend and, at least for cuelure, this attracts more females.     

Juvenile hormone biosynthesis and oocyte development in adult female Blattella germanica: Effects of grouping and mating

The roles of grouping and mating in modulating the activity of the corpora allata (CA) in adult female cockroaches were investigated using the in vitro radiochemical assay of juvenile hormone (JH) biosynthesis. Isolated virgin females have longer, asynchronous cycles of CA activity and oocyte maturation than do isolated females mated on day 8. Three factors were identified as the major contributors to this difference: (1) an experimental artifact of selection for sexually receptive females, (2) a positive effect of grouping on JH synthesis and oocyte maturation, and (3) a positive effect of copulation on oviposition and retention of the ootheca. Mated females constitute a subpopulation of receptive females that differ significantly from other females by having higher rates of JH synthesis prior to mating. The relative importance of such selection is substantial when the rate of mating is low, as in experiments with isolated females that are exposed to males for a short period of time. Long-term exposure of females to males introduces a grouping effect, which obscures any additional effect of mating on CA activity and oocyte development. However, mating influences ootheca formation and its retention. The effect of grouping can be mimicked in isolated females by transection of the nerves connecting the CA–corpora cardiaca complex to the brain, suggesting that in this insect isolation causes brain inhibition of the CA, and grouping provides disinhibitory stimuli that release the CA from brain inhibition.     

Acp70A regulates Drosophila pheromones through juvenile hormone induction

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. Here we demonstrate that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females.Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes.Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed.     

Hormonal and host factors stimulating pheromone synthesis in female western pine beetles, Dendroctonus brevicomis

ABSTRACT. Newly-emerged, unfed Dendroctonus brevicomis females produced large quantities of the pheromone exo -brevicomin when treated topically with the synthetic juvenile hormone 10,11-epoxyfarnesenic acid methyl ester (JH III). No exogenous source of pheromone precursor was required, and decapitation experiments showed that the synthetic JH was effective in the apparent absence of brain hormone. However, implantations of combined corpora allata—corpora cardiaca from either newly-emerged or fed (i.e. pheromone producing) females failed to stimulate exo -brevicomin synthesis by recipient unfed beetles. Biosynthesis of exo -brevicomin was induced in newly-emerged females by ingestion of host phloem, and the stimulatory phloem component was found in methanol extracts. Neither less polar solvent extracts of phloem nor artificial distension of the gut with air was effective in stimulating pheromone synthesis.     

Reproductive biology of the German cockroach,Blattella germanica: Juvenile hormone as a pleiotropic master regulator

Juvenile hormone (JH) exerts major pleiotropic effects on cockroach development and reproduction. The production of JH by the corpora allata (CA) in the adult female German cockroach, Blattella germanica, is dependent upon and modulated by both internal and environmental stimuli. Mating, intake of high-quality food, social interactions, and the presence of vitellogenic ovaries facilitate JH synthesis. Conversely, starvation, deficient diets, enforced virginity, isolation, and a pre- or post-vitellogenic ovary cause the CA to produce less JH. Sensory stimulation of the genital vestibulum by the ootheca also inhibits the CA via signals that ascend the ventral nerve cord. All these stimulatory and inhibitory signals are integrated by the brain, and a preponderance of favorable signals results in a graded lifting of brain inhibition, permitting the synthesis and release of JH. The effects of inhibitory signals on JH biosynthesis can be lifted experimentally by severing nervous connections between the brain and the CA. Such an operation accelerates activation of the CA. Besides controlling gonadal maturation in females, JH concurrently regulates the production of sexual signals, including both attractant- and courtship-eliciting pheromones, and the behavioral expression of calling (pheromone release) and sexual receptivity. Although JH is required for the expression of copulatory readiness in female B. germanica, it appears that signals associated with copulation (spermatophore, sperm, accessory secretions) can inhibit this behavioral state even when titers of JH are permissive for receptivity. These observations suggest that JH might regulate sexual receptivity in females indirectly through other directives. In males, JH accelerates not only the onset of sexual readiness but also synthesis of accessory reproductive products. Lastly, we present a novel cockroach control strategy that is based on the intimate association between food intake and rising JH titers in B. germanica females. JH analogs cause abortion of fertile oothecae in gravid females. In turn, rising JH titers and vitellogenic oocytes induce feeding in females. With strategic placement of insecticidal baits and JH analogs, gravid females, which normally feed little and are difficult to control, can thus be effectively targeted for elimination. Arch. Insect Biochem. Physiol. 35:405–426, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of juvenile hormone release and oocyte growth following (RS)-hydroprene treatment in virgin female Diploptera punctata

ABSTRACT. The effect of (flS)-hydroprene treatment (2, 20, 200 μ g) on JH release was assessed in virgin females of D. punctata (Eschscholtz) during the first 10 days of adult life as was basal oocyte length and number of cells in the CA. At a dose of 2 μ g hydroprene, JH release was stimulated slightly and, on days 4 and 6, oocyte growth was significantly greater than that of acetone-treated controls. A similar but more striking enhancement of JH release and basal oocyte growth was observed at a dose of 20 μ g and a significant inhibition of JH release, in concert with a rapid growth of basal oocytes, was observed at a dose of 200 μ g. During the observation period, the mean number of cells in the CA decreased in a dose-dependent fashion, with a highly significant reduction in numbers in 20 and 200 μ g-treated animals. Reimplantation of vitellogenic ovarioles (three or six) into ovariectomized virgin females also resulted in an enhancement of JH release; this indicates that virgin female CA can respond to the stimulatory action of the ovary and is consistent with a model for ( RS )-hydroprene action in which the 'positive feedback' effect (stimulation of JH release) observed with low doses of the analogue occurs as a consequence of the action of the analogue on the ovary. ( RS )-hydroprene treatment stimulates basal oocyte growth to the point at which the previously unstimulatory virgin oocytes are able to enhance JH release by a feedback loop involving the CA and probably the brain.