Responsiveness of honey bee (Apis mellifera L.) corpora allata to allatoregulatory peptides from four insect species

Five neuropeptides with known allatotropic or allatostatic activity in other insect species were examined for their effects on honey bee corpora allata. Using an in vitro radiochemical assay, we assessed the ability of these peptides to affect the biosynthesis of juvenile hormone III and its immediate precursor methyl farnesoate, as well as their effects on the conversion of methyl farnesoate into juvenile hormone. None of the allatostatins tested affected JH biosynthesis during the last larval instar of honey bee workers. Manduca sexta allatotropin, however, stimulated JH biosynthesis in a stage-specific and dose-dependent manner. Analysis of intraglandular contents of juvenile hormone and its precursor revealed that the allatotropin significantly increased JH precursor but did not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for early fifth-instar worker larvae. Studies also indicated that the allatotropic effect was reversible at the level of methyl farnesoate production.     

Regulation of juvenile hormone biosynthesis in Heliothis virescens by Manduca sexta allatotropin

In Heliothis virescens, reproduction is strictly dependent on juvenile hormone (JH). In females, mating induces a sharp increase in JH titers, which stimulates increased vitellogenin biosynthesis and higher rates of egg production. JH biosynthesis is presumably stimulated by production and/or release of stimulatory neuropeptides such as allatotropins. There is evidence that allatotropin of H. virescens may be structurally related to Manduca sexta allatotropin (Manse-AT). In a radiochemical in vitro assay, synthetic Manse-AT stimulated JH biosynthesis by corpora allata (CA) of virgin H. virescens females in a dose-dependent manner, but had no effect on CA activity in H. virescens males. In females, the CA showed a transient increase in sensitivity to Manse-AT shortly after mating. Several structurally related peptides stimulated CA activity to a similar extent as Manse-AT. Corpora allata activity was stimulated by a Ca2+ ionophore, A23187. A membrane-permeable Ca2+ chelator, BAPTA/AM, antagonized the stimulatory effects of Manse-AT, suggesting that Manse-AT may enhance CA activity by increasing intracellular Ca2+ concentration.     

Terminal stages in juvenile hormone biosynthesis in corpora allata of Diploptera punctata: Developmental changes in enzyme activity and regulation by allatostatins

The O-methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata, is a cytosolic enzyme. The activity of O-methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O-methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro. AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata: the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.     

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.     

Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia oleracea, and regulation by Manduca sexta allatostatin and allatotropin

Juvenile hormone (JH) biosynthesis and the effects of synthetic Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin (Mas-AT) were investigated in isolated corpora allata (CA) of Vth stadium larvae of the tomato moth, Lacanobia oleracea. Reversed-phase high-performance liquid chromatography (RP-HPLC) of JH extracted from CA shows that larvae produce predominantly JH II and its corresponding acid. It appears that the acid homologue is a result of JH esterase activity in the CA (and other tissues) rather than the lack of JH acid methyltransferase. Mean rates of synthesis (100-200fmol/pr/h) were inhibited ca. 70% by Mas-AS and stimulated in a dose-dependent manner up to three times by Mas-AT. However, Mas-AS had no significant effect on Mas-AT-stimulated rates of JH biosynthesis. Using RP-HPLC and an enzyme-linked immunosorbent assay (ELISA) to Mas-AT, a peak of Mas-AT-like immunoreactivity was detected in larval L. oleracea brain homogenates. Co-elution of this immunoreactive peak with synthetic Mas-AT suggests that this neuropeptide is also present in L. oleracea.     

Enhancement by farnesol and farnesoic acid of juvenile-hormone biosynthesis in induced low-activity locust corpora allata

Exogenous farnesol or farnesoic acid (FA) stimulates juvenile hormone III (JH III) biosynthesis by isolated corpora allata from Locusta migratoria in a dose-dependent manner. Farnesol and FA also stimulate a dose-dependent accumulation of substantial amounts of methyl farnesoate (MF), identified by gas chromatography-mass spectroscopy (GCMS) analysis, in the corpora allata. Lower quantities of MF were found in the incubation medium. Corpora allata, denervated 2 days prior to assay, showed low spontaneous rates of JH biosynthesis which were stimulated by farnesol and FA. The dose-response curves for control and denervated corpora allata were similar. During oocyte maturation the rate of farnesol and FA stimulation of JH biosynthesis increased gradually. However, after transection of nervus corporis allati 1 (NCA-1), the rate of stimulated JH synthesis was maintained at preoperative levels. Although the spontaneous rate of JH biosynthesis decreased rapidly after NCA-1 transection, denervated glands could still be stimulated by farnesol or FA to produce large amounts of JH. These results suggest that the low spontaneous rate of JH biosynthesis in denervated corpora allata is not caused by inhibition of the final steps of JH biosynthesis.     

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.     

Interaction between Manduca sexta allatotropin and manduca sexta allatostatin in the fall armyworm Spodoptera frugiperda

A peptide that strongly stimulates juvenile hormone (JH) biosynthesis in vitro by the corpora allata (CA) was purified from methanolic brain extracts of adult Spodoptera frugiperda. Using HPLC separation followed by Edman degradation and mass spectrometry, the peptide was identified as Manduca sexta allatotropin (Mas-AT). Treating the CA from adult S. frugiperda with synthetic Mas-AT (at 10(-6) M) caused an up to sevenfold increase in JH biosynthesis. The stimulation of JH synthesis was dose-dependent and reversible. Synthetic M. sexta allatostatin (Mas-AS) (10(-6) M) did not affect the spontaneous rate of JH secretion from CA of adult S. frugiperda, nor did any of the allatostatins of the Phe-Gly-Leu-amide peptide family tested. However, when CA had been activated by Mas-AT (10(-6) M), addition of synthetic Mas-AS (10(-6) M) reduced JH synthesis by about 70%. This allatostatic effect of Mas-AS on allatotropin-activated glands was also reversible. When CA were incubated in the presence of both Mas-AT (10(-6) M) and various concentrations of Mas-AS (from 10(-8) to 10(-5) M), the stimulation of JH-biosynthesis observed was inhibited in a dose-dependent manner. The experiments demonstrate a novel mechanism of allatostatin action. In S. frugiperda JH synthesis was inhibited only in those glands which had previously been activated by an allatotropin.     

Sex-specific developmental profiles of juvenile hormone synthesis in honey bee larvae

Summary Juvenile hormone synthesis in drone larvae of the honey bee was measured by an in vitro radiochemical assay. The developmental profile of corpora allata activity in male larvae showed considerable differences from queen larvae, the presumptive reproductive females, and was comparable to workers, the sterile female morph. Drone and worker larvae, however, differed drastically in the regulation of juvenile hormone biosynthesis, as revealed by the addition of farnesoic acid to the culture medium. This precursor stimulated juvenile hormone synthesis of drone glands nearly eightfold, whereas in worker larvae it is known to lead to an accumulation of methyl farnesoate. The sex-specific differences in endocrine activity indicate a role for juvenile hormone in the expression of genetically determined sexually dimorphic characters during metamorphosis, a role not currently accounted for in models describing endocrine regulation of insect development. Correspondence to: K. Hartfelder     

Allatotropin-like peptide in Heliothis virescens: tissue localization and quantification

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA.     

Manduca sexta allatotropin and the in vitro biosynthesis of juvenile hormone by moth corpora allata: a comparison of Pseudaletia unipuncta females from two natural populations and two selected lines

We compared the effects of Manduca sexta allatotropin (Manse-AT) on the rate of in vitro juvenile hormone (JH) biosynthesis by the corpora allata (CA) of different-aged virgin females from migrant (Quebec) and non-migrant (Azores) populations of the armyworm, Pseudaletia unipuncta, as well as from early- and late-calling lines selected from the Quebec population. There was a significant age x strain interaction, with the observed rates of JH biosynthesis in early adult life closely reflecting strain-specific differences in the age at onset of calling. In considering data for all ages combined, treatment of CA with Manse-AT resulted in a significant increase in the rate of JH biosynthesis for all but the Late strain, although significant differences for this strain were detected at certain ages. The CA of females from the Azores strain showed the strongest stimulation, with those of 0- and 1-day-old individuals displaying a singularly high degree of sensitivity. Selection for early- and late-calling lines resulted in significant differences in the temporal patterns of JH biosynthesis but did not markedly affect the sensitivity of the CA to Manse-AT. These findings are discussed within the context of the age-related differences observed in the rates of in vitro JH biosynthesis and JH haemolymph titers previously reported in comparisons of the Quebec and Azorean strains of the true armyworm.     

Caste Determination in Bombus terrestris: Differences in Development and Rates of JH Biosynthesis between Queen and Worker Larvae

To study the possible role of juvenile hormone in caste determination in Bombus terrestris, we measured development and rates of juvenile hormone biosynthesis in vitro in larvae destined to develop into either workers or queens. Larvae of both castes developed through four instars and had the same growth rates. However, the duration of the instars was longer for queen larvae, and their head width at the third and fourth instars was significantly larger. After validating the well-known radiochemical assay of JH for bumble bee larvae, we show that worker larvae corpora allata exhibited a constant and low rate of JH biosynthesis, never more than 5 pmol JH/h/pair. Queen larvae, in contrast, had two peaks of JH biosynthesis: a small one during the first instar, which has previously been correlated with caste determination; and a large peak, previously undetected, above 40 pmol JH/h/pair, during the second and third instars. We suggest that caste determination in this species is mediated by JH and that the duration of larval instars is a key factor. The possibility that the queen influences caste determination via an effect on instar duration is also discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Allatoregulatory peptides in Lepidoptera, structures, distribution and functions

Allatoregulatory peptides either inhibit (allatostatins) or stimulate (allatotropins) juvenile hormone (JH) synthesis by the corpora allata (CA) of insects. However, these peptides are pleitropic, the regulation of JH biosynthesis is not their only function. There are currently three allatostatin families (A-, B-, and C-type allatostatins) that inhibit JH biosynthesis, and two structurally unrelated allatotropins. The C-type allatostatin, characterised by its blocked N-terminus and a disulphide bridge between its two cysteine residues, was originally isolated from Manduca sexta. This peptide exists only in a single from in Lepidoptera and is the only peptide that has been shown to inhibit JH synthesis by the CA in vitro in this group of insects. The C-type allatostatin also inhibits spontaneous contractions of the foregut. The A-type allatostatins, which exist in multiple forms in a single insect, have also been characterised from Lepidoptera. This family of peptides does not appear to have any regulatory effect on JH biosynthesis, but does inhibit foregut muscle contractions. Two structurally unrelated allatotropins stimulate JH biosynthesis in Lepidoptera. The first was identified in M. sexta (Manse-AT) and occurs in other moths. The second (Spofr AT2) has only been identified in Spodoptera frugiperda. Manduca sexta allatotropin also stimulates heart muscle contractions and gut peristalsis, and inhibits ion transport across the midgut of larval M. sexta. The C-terminal (amide) pentapeptide of Manse-AT is important for JH biosynthesis activity. The most active conformation of Manse-AS requires the disulphide bridge, although the aromatic residues also have a significant effect on biological activity. Both A- and C-type allatostatins and Manse-AT are localised in neurosecretory cells of the brain and are present in the corpora cardiaca, CA and ventral nerve cord, although variations in localisation exist in different moths and at different stages of development. The presence of Manse-AS and Manse-AT in the CA correlates with the biological activity of these peptides on JH biosynthesis. There is currently no explanation for the presence of A-type allatostatins in the CA. The three peptide types are also co-localised in neurosecretory cells of the frontal ganglion, and are present in the recurrent nerve that supplies the muscles of the gut, particularly the crop and stomodeal valve, in agreement with their role in the regulation of gut peristalsis. There is also evidence that they are expressed in the midgut and reproductive tissues.     

Molecular characterisation of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sexta allatotropin and allatostatin preprohormone peptides

Allatotropin (AT) is a 13-residue amidated neuropeptide, first isolated from pharate adult heads of the tobacco hornworm, Manduca sexta (Manse-AT), which strongly stimulates the biosynthesis of juvenile hormones (JH) in the corpora allata (CA) of adult moths. In Spodoptera frugiperda, a cDNA that encodes 134 amino acids, including an AT peptide, has been cloned. The S. frugiperda allatotropin mature peptide (Spofr-AT) [GFKNVEMMTARGFa] is identical to that isolated from M. sexta. The basic organization of the Spofr-AT precursor is similar to that of Agrius convolvuli, M. sexta, Pseudaletia unipuncta, and Bombyx mori with 83-93% amino acid sequence identity. The Spofr-AT gene is expressed in at least three mRNA isoforms with 134, 171 and 200 amino acids, differing from each other by alternative splicing.All allatostatins (AS) have an inhibitory action on the JH biosynthesis in the CA. A cDNA that encodes 125 amino acid residues including one copy of the Manse-AS peptide has been cloned from S. frugiperda (Spofr-AS; QVRFRQCYFNPISCF). The basic organization of the Spofr-AS precursor is similar to that of P. unipuncta with 85% amino acid sequence identity.Using one step RT-PCR for semi-quantification of the gene expression, we showed that the three mRNAs of the Spofr-AT gene and the Spofr-AS gene are expressed in brains of last instar larvae, prepupae, pupae, and adults of both sexes of S. frugiperda with variable intensity.     

Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.     

Control of C-16 juvenile hormone biosynthesis in active corpora allata of female African locusts

Summary

Corpora allata from 8-day-old female Locusta migratoria, during the phase of yolk deposition, exhibit high rates of C-16 juvenile hormone (JH) biosynthesis. The effect of different potential factors which may be involved in the regulation of corpora allata activity is reported. The biosynthetic activity of corpora allata was determined by radiochemical assay.

In maturing females, no changes in corpora allata activity are detected during one daily cycle. Starvation reduces JH biosynthesis only 3 days after the beginning of the food deprivation. Suppression of the median neurosecretory material by electrocoagulation of the internal cardiaca tract (TCC-I) does not disturb JH biosynthesis whereas the transection of the allata I nerve fibres (NCA-I) or the electrocoagulation of the lateral neurosecretory pericarya results in a rapid decline of JH biosynthesis. These data indicate that the median and lateral allatotropins are different, and that only the lateral neurosecretory material exerts an allatostimulating action on corpora allata at the time of vitellogenesis. The corpora allata response to the median allatotropin changes during oocyte growth. C-16 JH and/or 20-hydroxyecdysone treatments in vitro (addition in the culture medium) and in vivo (injection in female) do not influence JH production in our experimental conditions.     

Juvenile hormone control of oögenesis in bumblebee workers, Bombus terrestris

In queenright workers of Bombus terrestris oögenesis is inhibited by the queen, the activity of the corpora allata is suppressed, and the resulting JH titer in the haemolymph remains low. In contrast, in queenless workers the JH production is stimulated on the first day of adult emergence, the JH titer increases, and eggs are rapidly formed. After injection of JH I in newly emerged workers oögenesis can also be induced in the presence of a queen in the same way as in queenless workers. The induced oögenesis is JH dosage dependent, dosages of less than 8 μg stimulate the production of vitellogenins, whereas a complete oögenesis can be induced by high dosages of about 50 μg. From studies on the rate of JH excretion it can be concluded that such high dosages must be injected to obtain the required JH titer during the whole egg maturation of 5 days. It is evident, therefore, that the initiation and the maintenance of oögenesis depends on JH.The injected JH ester is completely degraded to JH acid and JH diol mainly in the hindgut. The excretion starts quickly after the injection, but only traces of unchanged JH are excreted.From these results it can be suggested that a queen inhibits egg maturation in workers by suppressing the JH production in the corpora allata and thus lowering the JH titer. This influence also enables a queen to block oögenesis once stimulated, for instance in queenless workers. Breakdown of JH and excretion are not under the control of the queen and therefore neither of them play any rôle in regulating egg maturation in the worker caste.     

Juvenile hormone biosynthesis in moths: synthesis and evaluation of farnesol homologs as alternate substrates of farnesol oxidase

The oxidation of farnesol to farnesal is an important step in insect juvenile hormone (JH) biosynthesis and is mediated by one or more alcohol oxidases located within the minute endocrine gland, the corpus allatum. Because lepidopteran insects have the capacity to produce homologous JH structures, the substrate selectivity of farnesol oxidase was examined by determining the ability of several terpenol homologs to inhibit farnesol oxidation in moths. Results utilizing corpora allata homogenates from larval, adult, and embryonic Manduca sexta indicate that increased steric bulk at the C-3 position of the sesquiterpenol chain is detrimental to inhibitory potency. Triethylhomofarnesol (1h), which is precursor to JH 0 and therefore a physiologically important metabolite of M. sexta embryos, was found to be a poor inhibitor of farnesol oxidation but was oxidized in almost same amount as farnesol. This data indicate that farnesol oxidase of the corpus allatum plays a limited role in controlling JH homolog production in moths, and suggests that another oxidative enzyme, which is present at early stages of moth development, is involved in JH homolog construction.