Degradation of juvenile hormone and methylation of juvenile hormone acid by corpora cardiaca–corpora allata of the cockroach,Nauphoeta cinerea: I. Biochemical aspects

Corpora cardiaca-corpora allata (CC-CA) from vitellogenic females of Nauphoeta cinerea degraded, in vitro, racemic and (10R)-juvenile hormone III (JH III) at a rate of 249 pmol/CC-CA/h and 786 pmol/CC-CA/h, respectively. The major metabolite formed was JH III acid, together with some highly polar products. CC-CA homogenates degraded racemic JH III to a small extent, whereas (10R)-JH III was degraded efficiently to JH III acid. No highly polar products were formed by CC-CA homogenates. When CC-CA were incubated with racemic JH III acid, some of this substance was degraded to highly polar products, and a minor part was methylated to JH III. CC degraded very little JH III acid and did not methylate it to JH III. CC-CA homogenates methylated JH III acid very efficiently; we measured an apparent K_max of 37.8 μM and a V_max of 1,260 pmol/4 h/ CC-CA equivalent. The addition of JH III acid to CC-CA in vitro increased the rate of biosynthesis of JH III, as determined by measuring incorporation of methyl[¹⁴C]methionine into JH III. These data indicate that the metabolite JH III acid can enter the CA and be methylated to JH III.     

Pheromone, juvenile hormone, and social status in the male lobster cockroach Nauphoeta cinerea

In this study, the major pheromone component, 3‐hydroxy‐2‐butanone (3H‐2B), released by dominants was measured during early scotophase. Both the JH III titer in the hemolymph and the 3H‐2B content of the sternal glands of the dominants and subordinates were then measured during late scotophase and late photophase. These investigations were performed on encounter days 1, 2, 3, 5, 7, 9, 12, and 20. The results showed that, for non‐aggressive posture (AP)‐adopting socially naïve males (SNMs), both the 3H‐2B release and the hemolymph JH III titer were maintained at a low level. Once a fight occurred, 3H‐2B release was raised significantly in the AP‐adopting dominants, but not in non‐AP‐adopting subordinates, and remained raised throughout the entire experimental period. At 30 min after the first encounter, the hemolymph JH III titer was significantly increased in dominants, but not in subordinates. A significantly higher hemolymph JH III titer was observed in dominants during late scotophase on days 3, 5, 12, and 20 and during late photophase on days 3, 5, and 20. After fighting, the sternal gland 3H‐2B content of the dominants or subordinates was significantly lower than in SNMs. In dominants, the sternal gland 3H‐2B content during late scotophase was significantly lower than that during late photophase in the first 9 domination days, while, in the subordinates, the 3H‐2B content during late scotophase was either similar to, or significantly higher than, that in late photophase. In the dominants, 3H‐2B release and JH III titer were positively correlated. In rank switchers, the switched social status was positively correlated with both 3H‐2B release and JH III titer. Comparison of 3H‐2B release and JH III titer in 1‐time, 3‐time, or 5‐time dominants showed that, although winning significantly increased both 3H‐2B release and JH III titer, there is no significant difference in 3H‐2B release between 3‐ and 5‐time winners, while the JH III titer was most significantly increased in the 3‐time winners. The possible relationship between pheromone release, JH III titer, and social status is discussed. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley‐Liss, Inc.     

The isolation and identification of juvenile hormone from cockroach corpora allata in vitro

Methyl (2E, 6E) - 10, 11 - epoxy - 3, 7, 11 - trimethyl - 2, 6 - dodecadienoate (JH III) is synthesized by corpora cardiaca/allata of the American cockroach $\text{Periplaneta americana in vitro}$ and released into the culture medium. It constitutes the principal part of the biologically detectable JH activity. A fully synthetic medium without terpenoid precursors or serum was used, which proves, that the entire molecule is synthesized $\text{de novo}$. The presence of JH III in orders as different as Blattodea, Orthoptera, Coleoptera and Lepidoptera indicates, that it is the most widely occuring JH of insects.     

Inhibition of juvenile hormone III biosynthesis in cockroach corpora allata by interference with the S-adenosylmethionine-dependent transmethylation

The synthesis of juvenile hormone-III by corpora allata of the cockroach Diploptera punctata is dependent under in vitro conditions upon a supply of exogenous methionine. Radiolabelled S-adenosylmethionine was identified by HPLC in extracts of corpora allata incubated with either [methyl-³H]methionine or [³⁵S]methionine. Juvenile hormone (JH) synthesis by intact glands in vitro was inhibited by cycloleucine and selenomethionine, but this inhibition could be relieved by increasing the concentration of methionine. S-adenosylhomocysteine or sinefungin had little or no inhibitory effect on JH synthesis by intact glands, but 5′-deoxy-5′-methylthioadenosine was inhibitory. Adenosine and homocysteine synergistically inhibited JH synthesis. These results show that JH-III synthesis by intact glands can be inhibited by interfering with the S-adenosylmethionine-dependent transmethylation, and suggest that the product and inhibitor of that reaction, S-adenosyl-homocysteine, is rapidly hydrolyzed to adenosine and homocysteine in the corpora allata.     

The effect of l-methionine concentration on juvenile hormone biosynthesis by corpora allata of the cockroach Diploptera punctata

The effect of varying l-methionine (l-met) concentration on rates of juvenile hormone (JH) biosynthesis/release by corpora allata of females of the viviparous cockroach Diploptera punctata has been studied using a radiochemical assay. Both high activity glands (corpora allata from day 5 females) and low activity glands (corpora allata from day 11 females) were used to study the dose dependence of JH biosynthesis on l-met concentrations, under both de novo (spontaneous) conditions of JH biosynthesis and stimulated conditions (in the presence of the exogenous JH III precursor farnesoic acid). Maximal rates of JH biosynthesis/release were observed at l-met concentration of 20 μM (spontaneous) and 40 μM (stimulated). Below these concentrations, rates of JH biosynthesis declined linearly with decreasing l-met concentration. Optimal concentration of l-met appeared to be similar for both high and low activity corpora allata, under spontaneous and stimulated conditions of biosynthesis. Above 40 μM l-met, no increase in rates of JH biosynthesis was observed. It appears that the corpora allata of D. punctata are efficient scavengers of l-met and are able to utilize even low concentrations of the substrate for JH biosynthesis. The corpora allata of D. punctata may prove useful for the biosynthesis of authentic JH III, radiolabelled in the methyl position using as methyl donor, l[methyl-³H]met of high specific activity.     

Regulation of sex pheromone production in the male Nauphoeta cinerea cockroach: Role of brain extracts,corpora allata�(CA), and juvenile hormone (JH)

The neuroendocrine mechanisms underlying pheromone regulation in cockroaches are unclear because of a lack of physiological and chemical data. The present report describes experiments designed to determine the role of the brain, corpora allata, and juvenile hormone III in the production of sex pheromone by male Nauphoeta cinerea cockroaches. The levels of two sex pheromone components, i.e., acetoin and 2‐methylthiazolidine, were measured by gas chromatographic analysis of sternal gland extracts obtained from individual males. Allatectomy or decapitation performed up to 2 to 3 days after imaginal molt caused a decrease in sex phermone levels. Conversely decapitation or allatectomy performed after 3 to 4 days post‐eclosion had almost no effect on sex pheromone levels. Injection of JHIII into allatectomized and decapitated males stimulated pheromone production while injection of brain extract had no effect. These results indicate that JHIII is involved in the differentiation of sternal glands and that regulation via pheromone biosynthesis activating neuropeptide (PBAN) does not occur in N. Cinerea cockroaches. Arch. Insect Biochem. Physiol. 40:165–172, 1999. © 1999 Wiley‐Liss, Inc.     

Cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach, Diploptera punctata

The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates insect growth, metamorphosis and reproduction. In the corpus allatum (CA) of the female adult cockroach Diploptera punctata, cyclic rise and decline in JH synthesis rates occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increase with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) is low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP content. Consequently, elevated cGMP may inhibit protein synthesis or trigger massive and synchronous autophagic activities, resulting in cell atrophy and reduction of protein content. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis.     

Ultrastructural localization of juvenile hormone biosynthesis by insect corpora allata

Summary The conversion of exogenous ³H-farnesenic acid to ³H-methyl farnesoate and ³H-C₁₆ juvenile hormone (JH) has been followed in the corpus allatum (CA) cells of the desert locust Schistocerca gregaria by means of electron microscopic autoradiography. Aerobic and anaerobic chase incubations have been used to modify the quantities of these three compounds within the CA cells. Under all incubation conditions, radiolabel is found associated almost exclusively with the subcellular membrane systems — smooth endoplasmic reticulum (SER) and Golgi elements —and with the mitochondria. CA cells are probably similar to vertebrate steroid-synthesizing cells in that the secretory product is synthesized in the SER and mitochondria.Radiolabel was found to be present in all cells of the CA suggesting that all cells are capable of at least the final two stages of JH biosynthesis (the esterification and epoxidation of ³H-farnesenic aid). This indicates that JH biosynthesis may be regulated through changes in the biosynthetic capabilities of individual cells rather than through changes in the total number of cells engaged in biosynthesis. Radiolabel was not observed to be associated with any distinctive cellular product, a result which provides additional evidence for the suggestion that the release of JH from the CA is governed by laws of simple physical diffusion.Supported by operating grants from the National Research Council of Canada to SST and ASMS. ³H-farnesenic acid was supplied by the late Dr. A.F. White of the Unit of Invertebrate Chemistry and Physiology, A.R.C., University of Sussex. We thank Dr. G.E. Pratt for helpful discussions     

Development changes in juvenile hormone and juvenile hormone acid titers in the hemolymph and in-vitro juvenile hormone synthesis by corpora allata of the silkworm,Bombyx mori

A simple method was developed to quantify hemolymph juvenile hormone (JH) and JH acid in hemolymph extracts from Bombyx mori with an established radioimmunoassay (RIA) for JH I. When various organic solvent extracts of hemolymph were assayed by RIA, levels of non-specific binding of the labeled ligand in the assay were determined to be greater than 50% of the maximum amount of the label bound by the antiserum. When hemolymph was diluted with methanol:water:8.4N ammonium hydroxide (10:9:1) and extracted with isooctane, non-specific binding was only 50% higher than control levels obtained with the assay buffer alone. The organic phase contained only JH and aqueous phase, JH acid. Consequently, this extraction method was used to prepare samples for RIA and enabled the separate measurement of JH and JH acid in hemolymph. With this method, changes in the hemolymph titers of JH and JH acid were determined from the third instar through early pupal stage of Bombyx mori. Changes in the in vitro secretory activity of corpora allata and brain-corpora cardiaca-corpora allata complexes from fifth instar larvae were also determined by using JH I RIA of the incubation medium.     

Methyl farnesoate and juvenile hormone III in normal and precocene treated embryos of the ovoviviparous cockroach Nauphoeta cinerea

Summary

Titers of juvenile hormone III and methyl farnesoate, its unepoxidized precursor, were measured throughout embryonic development using a gas-chromatographic method and it was revealed that both substances are undetectable before dorsal closure. Thereafter they both reach similarly high concentrations (800 ng/g) until a few days before hatching, when their titers begin to decrease. Application of the precocene analogue, 7-ethoxy-6-methoxy-2,2-dimethyl-chromene, to egg-cases at dorsal closure results in very low or undetectable titers of juvenile hormone III, depending on the dose applied, whereas methyl farnesoate is seen to reach high levels similar to those seen in the controls. The severe developmental disturbances observed suggest that juvenile hormone III is very important for normal development and formation of the 1st instar larva.     

20-hydroxyecdysone stimulation of juvenile hormone biosynthesis by the mosquito corpora allata

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or −24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the −24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the −24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.     

Inhibition of juvenile hormone III biosynthesis by fluoromevalonolactone and citronellyl phenyl imidazoles in isolated corpora allata from the cockroach Periplaneta americana

1-Citronellyl-5-phenyl imidazole (1,5-CPI), 1-citronellyl-4-phenyl imidazole (1,4-CPI) and 1-citronellyl-2-phenyl imidazole (1,2-CPI) were tested as inhibitors of JH-III biosynthesis in vitro. 1,5-CPI was found to be most active followed by 1,2-CPI. The least active isomer was 1,4-CPI. Inhibition of JH biosynthesis by 1,5-CPI resulted in no significant accumulation of the epoxidation substrate methyl farnesoate, and piperonyl butoxide, a known microsomal epoxidase inhibitor, produced only a slight increase in methyl farnesoate. Topical application of fluoromevalonolactone resulted in reduced biosynthetic capability of subsequently excised corpora allata.     

Gas chromatography-mass spectroscopy analysis of juvenile hormone released by insect corpora allata

Corpora allata incubated in appropriate medium release several compounds including juvenile hormones. Juvenile hormones (14C labeled or unlabeled) were extracted with hexane and directly analyzed by gas chromatography-mass spectroscopy. This method allowed the qualitative and quantitative analysis of total released juvenile hormone. It could also be used as a routine assay for evaluation of corpus allatum activity. Data obtained by this method were compared to those obtained by radiochemical assay.     

The effects of octopamine on juvenile hormone biosynthesis,electrophysiology, and cAMP content of the corpora allata of the cockroach Diploptera punctata

Summary Juvenile hormone production by the corpora allata of the adult female cockroach, Diploptera punctata, can be modulated by treatment with the biogenic amine, octopamine. Endogenous octopamine has been identified within the CA, using HPLC and electrochemical detection. Treatment with octopamine results in a sinusoidal, dose-dependent inhibition of JH biosynthesis by CA from day 2 virgin females, with maximal inhibition occurring at 10^-10 M and 10^-4 M. In day 4 and day 8 mated female corpora allata octopamine inhibited JH biosynthesis at 5·10^-5 M. Although the elevation of either cAMP or cGMP within the CA is known to be associated with an inhibition of JH biosynthesis, treatment with high concentrations of octopamine results in an increase in the level of cAMP but not cGMP. This effect is both dose- and time-dependent.Octopamine treatment also initiates changes in the passive membrane responses of the CA. Superfusion of CA with octopamine results in a pronounced hyperpolarization of CA cells and an increase in the electrotonic potential (indicative of the degree of electrical coupling between CA cells). This effect could be blocked by the octopamine receptor blocker phentolamine. Treatment with octopamine or phentolamine also blocked the hyperpolarization of CA cells normally associated with electrical stimulation of the axon tracts innervating the CA.We hypothesize that octopamine may be a natural neuromodulator of JH production by CA, regulating ion channels in CA cells themselves as well as release of the inhibitory neuropeptide, allatostatin, from the terminals within the CA.Abbreviations 4-AP 4-aminopyridine - CA corpora allata - CC corpora cardiaca - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid - HPLC high pressure liquid chromatography - IBMX 3-isobutyl-1-methylxanthine - JH juvenile hormone - ms millisecond - nA nanoampere - NCA I nervi corporis allati I - OCT octopamine - TEA tetraethyl ammonium     

Ultrastructural changes in corpora allata during a cycle of juvenile hormone biosynthesis in embryos of the viviparous cockroach, Diploptera punctata

We have observed changes with time in the fine structure of corpora allata (CA) during a known cycle of increasing and decreasing juvenile hormone (JH) synthesis in late embryos of Diploptera punctata. A previous report showed that rates of JH release were relatively low in 28-day-old embryos, but CA activity subsequently rose linearly to a peak on about day 42, and thereafter steadily declined to a low level on day 64 just before birth (Holbrook et al., 1997). We now show that, regardless of rate of JH synthesis, CA cells are large and replete with organelles which nevertheless exhibit variable morphology in embryos of different age. Highly active CA cells on day 40 contain abundant ring-form mitochondria, whereas CA cells of low activity on days 28 and 64 contain mitochondria that are rod-shaped or globular. Mitochondrial cristae were scarce and indistinct on day 28 but numerous and well developed on day 64. Endoplasmic reticula (ER) are rare on day 28 and appear in increasing numbers when CA activity rises. On day 40, ER are abundant and often exhibit a whorl-like appearance which is not observed on day 28. After day 44, when biosynthetic activity is declining, whorls of ER gradually decrease in number and are ultimately replaced by vesicular smooth ER on day 64. Neurosecretions are found only after day 38, by which time rates of JH synthesis have increased substantially from those of day 28. Except for membranous autophagic vacuoles, which are frequently found when ER whorls disintegrate as rates of JH synthesis decline toward birth, most autophagic vesicles such as multivesicular vesicles and dense bodies occur only sporadically among CA cells at all examined ages. We conclude that synchronous autophagy of exhausted organelles, which results in atrophy of CA cells and long-term arrest of JH synthesis in adult females of D. punctata, does not occur in embryos. The slow cyclic change in rate of JH synthesis in embryonic CA is most likely due to asynchronous autophagic activity and to alterations in certain unique features of intracellular organelles.     

A stage-specific ovarian factor with stable stimulation of juvenile hormone synthesis in corpora allata of the cockroach Diploptera punctata

This is a study of a feedback loop from a stimulated organ to glands that produce the stimulatory hormone in the cockroach Diploptera punctata. In this insect as in many others, juvenile hormone (JH) produced by corpora allata (CA) stimulates vitellogenesis. In our previous studies, transplantations of ovaries at certain stages of development into ovariectomized mated females stimulated JH synthesis within 24h. An in vitro study by other investigators showed that all stages of ovaries release a stimulatory factor into culture medium that was not retained on a solid-phase extraction column but occurred in the aqueous flow-through. The present study is a comparison of the effect of medium conditioned with ovaries from days 1-4 and 8 of the first reproductive cycle, to the effect of the flow-through of that medium on members of a pair of CA from day 3 females. Results provide evidence for an ovarian factor that stimulates JH synthesis by CA in vitro after removal from the conditioning medium (i.e., stable stimulation). Only medium conditioned with ovaries from days 2 or 3 females significantly stimulated CA more than flow-through. Stimulation was dose dependent, sensitive to trypsin, and survived freezing. These results indicate that CA can be directly and stably stimulated by a stage-specific peptidergic ovarian factor.     

An allatostatic factor and juvenile hormone synthesis by corpora allata in Locusta migratoria

The hemolymph juvenile hormone (JH) titer in sexually immature female adults of Locusta migratoria (Ibaraki strain, Japan) was lower than in sexually mature females; nevertheless, JH synthetic activity by the corpora allata (CA) in vitro was considerably higher in immature females than in sexually mature females ([Okuda et al., 1996]). We carried out experiments to explain this contradiction. The CA activity of sexually immature female adults was very low when the CA were incubated as a complex together with the corpora cardiaca (CC) and brain. When the same complex was assayed after cutting the nerve cord connecting the CC and CA (NCA1), JH synthesis by the CA was enhanced tenfold. When this pair of CA was incubated in fresh medium without the CC and brain, JH synthesis was further increased. Therefore, the higher in-vitro JH production by CA from immature female adults was the result of isolation of the CA from the brain and CC. A methanolic extract of brain-CC complexes contained a factor that inhibited JH synthetic activity by CA in vitro in both immature and mature insects, and this inhibition was reversible. The factor was heat-resistant but lost allatostatic activity after pronase digestion. These results indicate that the allatostatic factor is probably a heat-stable peptide.     

Farnesoic acid stimulation of C16 juvenile hormone biosynthesis by corpora allata of adult female Diploptera punctata

Biosynthesis of C₁₆ juvenile hormone (C₁₆JH) by isolated corpora allata (CA) of the viviparous cockroach Diploptera punctata has been studied by a radiochemical assay. This assay uses the incorporation from methyl labelled l-methionine in the methyl ester moiety of C₁₆JH. The optimal concentration of l-methionine in the medium has been determined. When varying proportions of (methyl-³H)- and (methyl-¹⁴C)-labelled l-methionine are used in the assay, no isotope effect was observed indicating that labelled l-methionine can be used as a mass marker to quantify C₁₆JH synthesis in D. punctata. C₁₆JH synthesis was stimulated by addition of farnesoic acid (FA) to the medium (maximum at 30–40 μM). When stimulated with 30 μM FA, the C₁₆JH release rate was directly dependent on C₁₆JH synthesis rate, and no intraglandular accumulation of either C₁₆JH or its immediate precursor methyl farnesoate were observed. Spontaneous and FA-stimulated rates of C₁₆JH release were studied during the first reproductive cycle of the adult female. The two final steps of C₁₆JH biosynthesis are not physiologically rate-limiting, as the ratio of spontaneous to FA-stimulated C₁₆JH release (fractional endocrine activity ratio) is always lower than unity. There is a precise relationship between the length of the basal oöcytes and the rate of C₁₆JH release.     

The possibility of sex and nymph discrimination by the male cockroach,Nauphoeta cinerea

The possibility of sex and nymph discrimination by males was investigated in the cockroach,Nauphoeta cinerea (Olivier). A sexually mature male takes a courting position toward a sexually mature female when he comes into contact with her and recognizes her through antennal contact. In contrast, males often behave aggressively toward each other: they bite at each others; wings and/or legs, chase each other and antennate mutually. The male, however, does not show conspicuous behavior (mating behavior or aggressive behavior) toward nearby nymphs. The male produces audible sounds when he courts a sexually mature but non-receptive female who does not respond to his courtship behavior. We found that the male also stridulates after he repeatedly courts immature teneral females, males and (last-instar) nymphs. After the bodies of teneral insects are sclerotized, the male shows courship and stridulation behavior toward sexually mature but non-receptive females but not toward mature males and nymphs. At this stage the male begins to behave aggressively toward other post-teneral mature males. We think that the variability of the sexually mature male's behavior toward other conspecifics (courtship behavior toward female, aggressive behavior toward male and no conspicuous response toward nymph) results from the male's recognition of adult and nymph.