Proctolin: A possible releasing factor in the corpus cardiacum/corpus allatum of the locust

The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10(-8)M and maximal release at about 10(-7)M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (<35 pmol h(-1) per CA). In contrast, proctolin did not alter the synthesis and release of JH from the cockroach CA. These results suggest that proctolin may act as a releasing factor for AKHs and JH in the locust but does not act as a releasing factor for JH in the cockroach.     

Action of a juvenile hormone analogue on the activity of Periplaneta americana corpora allata in vitro and on juvenile hormone III levels in vivo

The effects of the juvenile hormone (JH) analogue fenoxycarb (ethyl[2-(4-phenoxyphenoxy)-ethyl]carbamate) on the activity of corpora allata (CA) from adult female Periplaneta americana have been investigated. The in vitro biosynthesis of JH III by isolated CA was inhibited by about 85% in the presence of a high concentration (1 × 10⁻⁴ M) of fenoxycarb. However, at lower concentrations (1 × 10⁻⁶ M and 1 × 10⁻⁸ M) no inhibition of JH biosynthesis was apparent. Topical treatment of adult female cockroaches with fenoxycarb (100 μg/insect) did not reduce the subsequent rate of JH III biosynthesis by CA in vitro. By contrast, the same treatment markedly reduced the titre of endogenous JH III in intact cockroaches. These results suggest that CA activity in adult female P. americana may be controlled by negative feedback, and that this system of control is dependent on the maintenance of contact between the CA and nervous or humoral factors in the intact insect. Alternatively, it is possible that treatment with fenoxycarb increases the rate at which endogenous JH is metabolized.     

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.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

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.     

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     

Biosynthesis and regulation of juvenile hormone III,juvenile hormone III bisepoxide,and methyl farnesoate during the reproductive cycle of the grey fleshfly,Neobellieria (Sarcophaga) bullata

To study the effect of brain signals on the biosynthesis of juvenile hormone by the corpora allata of the grey fleshfly Neobellieria bullata, exposed corpora allata connected to the brain were surgically removed from sugar-fed flies and incubated in vitro with L -[³H-methyl]methionine. After incubation, the media together with the tissues were analyzed by HPLC. [³H]Juvenile hormone III (JH III), [³H]JH III bisepoxide (BE), [³H]methyl farnesoate (MF) and an unknown [³H]labeled metabolite (Un) were identified as the primary products. The rate of synthesis of [³H]JH III bisepoxide was higher than that of [³H]JH III, [³H]MF and [³H]Un. Two days after a liver meal, female flies synthesized more JH III, MF, BE, and the Un than did males. Synthesis of JH III, BE, and MF in females was lower during the previtellogenic, sugar-feeding period than during the vitellogenic liver-feeding period. Isolated corpus cardiacum–corpus allatum (CC-CA) complexes that were incubated in vitro synthesized less JH III, MF, and BE, as compared to complexes that were attached to the brain, indicating that the brain probably modulates the biosynthesis of JH III, MF, and BE in the corpora allata. Upon incubation of brain–CC–CA complexes with Neb-TMOF (10^–8 M), Neb-colloostatin (10^–8 M), ovarian, or brain extracts resulted in significant inhibition of JH III and BE biosynthesis in the presence of ovarian extracts. These results indicate that allatostatin-like factors are present in the ovary of the flesh fly. Arch. Insect Biochem. Physiol. 37:248–256, 1998. © 1998 Wiley–Liss, Inc.     

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.     

Biosynthesis and degradation of juvenile hormone in corpora allata and imaginal wing discs of Galleria mellonella (L.)

The rate of juvenile hormone (JH) biosynthesis by corpora allata-corpora cardiaca complex (CA/CC) during two last larval instars of Galleria mellonella was analysed. The rate of biosynthesis reaches maxima at the beginning of the VIth and VIIth instars. It is markedly reduced before the last larval ecdysis and after the first day of the last larval instar. After passing the second day of the last larval instar CA/CC exhibits again an increased ability for the biosynthesis of JH.The JH esterase activity in CA/CC is very low at the beginning of last larval instar and rapidly increases after the first day of this instar. Beginning on the second day of last larval instar the rate of JH hydrolysis is always higher than the rate of JH synthesis in CA/CC. It is concluded that the secretion of JH by CA/CC is possible until the second day of the last larval instar. After this, JH-acid can be supplied by CA/CC to peripheral tissues.The imaginal wing discs of mobile prepupa exhibit the ability to methylate JH-acid. It is concluded that some elevations of JH titre in G. mellonella haemolymph after the second day of VIIth instar are due in part to JH-acid methyltransferase activity in the imaginal discs.     

Changes in juvenile hormone synthesis in the termite Reticulitermes flavipes during development of soldiers and neotenic reproductives from groups of isolated workers

Workers of Reticulitermes flavipes were isolated in groups of increasing numbers to determine the in vitro rates of juvenile hormone (JH) synthesis by individual pairs of corpora allata (CA) as other castes differentiated. Only neotenic reproductives developed in groups of 12. Mean JH synthesis rates increased after 5 weeks but only a few individuals had significantly higher rates, about 0.4 pmol/pair/h, which occurred at about 3 weeks before neotenics developed. Soldiers and neotenics developed in groups of 50. Mean rates increased to a peak at week 6 after isolation, but only a few individuals had rates approaching 1 pmol/pair/h, which occurred at the same time after isolation as the development of pre-soldiers. JH synthesis by CA of pharate pre-soldiers and soldiers was low compared to that of pharate workers and neotenics. CA of pre-soldiers attained a peak mean rate of JH synthesis of 0.9 pmol/pair/h at 6 days of age, whereas CA of soldiers attained only a peak mean rate of 0.3 pmol/pair/h. These measurements of JH synthesis by individual pairs of CA suggest that the few workers destined to become pre-soldiers have 2.5-fold higher JH synthesis than the few that would develop into neotenic reproductives, and show that a cycle of synthesis accompanies the development of pre-soldiers into soldiers.     

Juvenile Hormone biosynthesis and release during oocyte development in Phormia regina Meigen

A radiochemical assay for Juvenile Hormone (JH) biosynthesis and release by the corpus allatum (CA) was used to assess the effects of diet on CA activity of adult female Phormia regina (Meigen) fed either sugar-water or sugar-water-liver. CC-CA complexes were incubated in L-methionine-free medium 199 supplemented with ³H-L-methionine. The rate of JH release by the CC-CA complexes is linear for 3 h and declines slightly thereafter. JH III appears to be one of the major components of the isooctane-extractable product from incubated CC-CA. High pressure liquid chromatographic analysis indicates that 10% of the released radiolabelled product is JH III. Rates of JH release show a strict dependence on L-methionine concentration in the incubation medium, with optimal rates occurring between 100 and 150 μM L-methionine. JH release is at a low level (<0.02pmolh^-1 per pair of CC-CA) in flies fed only sugar-water, but increases dramatically in flies fed sugar-water-liver (average release rate of 0.2pmolh^-1 per pair of CA, 24h after a liver meal). The rate of JH release increases steadily to more than 1.2pmolh^-1 per pair at 128h of age (i.e. 56h after a liver meal) at which time oocytes are mature. Elevated rates of JH release in vitro appear to be correlated in vivo with the appearance of vitellogenin in the haemolymph and its uptake by the developing oocytes.     

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.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. 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 would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Expression of juvenile hormone acid O‐methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone (JH), a sesquiterpenoid synthetized by the insect corpora allata (CA), plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O‐methyltransferase (JHAMT). Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica (L.) (BgJHAMT). BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3′‐UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi‐triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body vitellogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin (TOR). Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH‐specific branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.     

Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata,and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.     

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.     

Changes in the size of corpora allata,in the juvenile hormone III titer in the hemolymph,and in the protein content of terminal oocytes throughout the first gonadotropic cycle in Aiolopus thalassinus (Insecta: Orthoptera: Acrididae)

In Aiolopus thalassinus (Fabr.), the changes in the volume of the corpora allata (CA), in the concentration of juvenile hormone III (JH III) in the hemolymph, and in protein content in the terminal (t) oocytes were studied during the first gonadotropic cycle. These parameters could be better related to the volume of t-oocytes than to age after emergence. The JH III titer curve was maximum (2.9 pmol/10 μl) at an oocyte volume of 1.2 mm³. Before oviposition (days 10–16) the JH III titer decreased to 0.65 pmol/10 μl hemolyph. The increase in JH III titer reflected a period of high protein storage in the t-oocytes. The largest volume of the CA was reached at the beginning of yolk storage in the t-oocytes. The highest JH III titer did not correspond with the largest volume of CA, which occurred much earlier. © 1993 Wiley-Liss, Inc.     

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.     

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

In vitellogenic females of Nauphoeta cinerea, injected (10R)-juvenile hormone (JH) III was degraded more rapidly than racemic JH III: we measured a half-life of 21 min (with or without coinjection of lipophorin) for the former and 24 min (with coinjection of lipophorin) and 43 min (without coinjection of lipophorin) for the latter. One to two hours after injection, JH III acid was the major metabolite observed; in addition, several highly polar products were found. The half-life of injected racemic JH III acid was 19 min with coinjection of lipophorin and 4 min without. The JH III acid titer in hemolymph was low (around 5–10 pmol/ml) in last instar larvae and previtellogenic and pregnant females and reached higher values (40–100 pmol/ml) in vitellogenic and ovulating females. Racemic JH III acid could be methylated in vitro to JH III by corpora cardiaca–corpora allata (CC-CA) from penultimate instar larvae and females at stages between adult ecdysis and ovulation and at the very end of pregnancy, but not by CC-CA from last instar larvae and adult females at earlier stages of pregnancy. This indicates that CC-CA are capable of methylating JH III acid only at stages when JH III is detectable in the hemolymph. In double-labelling experiments with CC-CA from vitellogenic females and L-[¹⁴C]methionine and [³H]JH III acid as precursors, we observed that only a small proportion (1–8%) of total biosynthesized JH III was derived from JH III acid when the latter was present at physiological concentration. This suggests that in vivo recycling of JH III acid by CC-CA plays only a minor role in the regulation of the titer of JH III and JH III acid.     

Biological effects of synthetic AKH in Manduca sexta and estimates of the amount of AKH in corpora cardiaca

Dose-response curves were measured with synthetic Manduca adipokinetic hormone (AKH) for glycogen phosphorylase activation in larvae and for lipid mobilization in adults. Both responses are known hormonal functions in Manduca sexta. In ligated larvae, full activation of glycogen phosphorylase was achieved with 0.1 pmol and half-maximal activation with 0.03-0.04 pmol. Maximal lipid mobilization in adults required 10 pmol and half-maximal mobilization 0.15 to 0.2 pmol, respectively. An estimate of AKH content of corpora cardiaca from M. sexta was gained by comparing the dose-response curves for synthetic Manduca AKH with curves from gland extracts. Corpora cardiaca extracts were also quantitated by high performance liquid chromatography. According to both estimates corpora cardiaca of adults contain 10-20 pmol AKH per pair, while a pair of larval corpora cardiaca contains 0.7-2 pmol.