Cardiaca-corpora Allata Research Articles

Quantitative Determination of Myotropic Neuropeptide in the Nervous System of the Cockroach Leucophaea Maderae: Distribution and Release of Leucokinins

ABSTRACT Being interested in the distribution and function of insect myotropic peptides, we developed a sensitive radioimmunoassay (RIA) using an antiserum directed against the cockroach neuropeptide leucokinin I. The levels of leucokinin immunoreactivity were measured in extracts of different portions of the nervous system, in the retrocerebral complex and in the hemolymph of the cockroach Leucophaea maderae. The brain contains about 1.9pmol of immunoreactive material while the ganglia of the ventral nerve cord each contain less than 10% of this amount. Large amounts of leucokinin immunoreactivity (6.6pmol per tissue) were found in the corpora cardiaca–corpora allata (CC–CA) complex and in the hemolymph the titer was in the nanomolar range. When levels of leucokinins were compared in male and female cockroaches, no differences could be detected in any of the sample tissues. A calcium-dependent release of leucokinin-immunoreactive material could be induced from isolated CC–CA complexes by high-potassium depolarization. The amount of released immunoreactivity, as measured in the bathing saline by RIA, was about 2% of the extractable leucokinin immunoreactivity in the CC–CA complex. In Leucophaea maderae, the leucokinins apparently act both as neuroactive substances in the central nervous system and as neurohormones released into the circulation.

Age dependency and regulatory properties of juvenile hormone III biosynthesis in adult male crickets, Gryllus bimaculatus

The in vitro rate of juvenile hormone III (JH III) biosynthesis by corpora cardiaca-corpora allata complexes from paired and unpaired adult males of Gryllus bimaculatus is age-dependent. Both groups display a peak of juvenile hormone III production on day 2 after the imaginal moult, with the peak rate in paired males reaching about double that of unpaired animals. The juvenile hormone III synthesis then declines steadily in unpaired males, while in paired males additional phases of elevated rates occur. The juvenile hormone III titre in the haemolymph (as measured by radioimmunoassay) parallels the biosynthesis rate of juvenile hormone III for the first 4 days of adult life. Stimulation of the corpora allata with 200 μM farnesol in vitro produces no increase in juvenile hormone III production, but methyl farnesoate accumulates by a factor of 17 times over control values. Elevated Ca 2+ concentrations in the incubation medium stimulate the juvenile hormone III synthesis in vitro in a dose-dependent manner, while increased K + concentrations inhibit synthesis almost completely. Combined treatment with elevated Ca 2+ and exogenous application of 20 μM farnesol indicates no synergistic increase in methyl farnesoate accumulation, even when free calcium is increased 20-fold. Elevated K + combined with 200 μM farnesol leads to a stimulation of juvenile hormone III biosynthesis and especially methyl farnesoate accumulation. Male corpora allata, completely deprived of nervous connections during incubation or incubated together with detached brain or as intact brain-endocrine complexes, always show lower biosynthetic activity compared to control CC-CA complexes.

Differential degradation of racemic and 10R-juvenile hormone III by cockroach ( Nauphoeta cinerea) haemolymph and the use of lipophorin for long-term culturing of corpora allata

Degradation of racemic and 10R-juvenile hormone III was analysed in warm and cold bled haemolymph and after fractionation in a KBr gradient with two assay systems. In the partition assay, where highly diluted haemolymph or haemolymph fractions were incubated with high substrate concentrations, racemic juvenile hormone III was metabolized much more rapidly than 10R-juvenile hormone III, particularly when warm bled haemolymph was used. This suggests the existence of enzymes which preferentially degrade 10S-juvenile hormone III and their induction or activation by warming. In another assay, juvenile hormone III degradation was measured in undiluted or only slightly diluted haemolymph or fractions thereof with physiological substrate concentrations. In this assay only minor quantitative differences were seen between racemic and 10R-juvenile hormone III and both enantiomers were degraded much more efficiently in cold bled haemolymph than in warm bled haemolymph. The major metabolite formed comigrated on thin-layer chromatography with juvenile hormone III acid, but with cold bled haemolymph also highly polar metabolite(s) comigrating with juvenile hormone III acid-diol were seen. This latter metabolite was more abundant when 10R-juvenile hormone III was used as substrate. Lipophorin purified by KBr gradient centrifugation was shown to bind 10R-juvenile hormone III with a high affinity ( K d of 4.08 ± 1.0 nM). When lipophorin was added to culture medium of corpora cardiaca-corpora allata production of juvenile hormone III in vitro continued over several days; in the absence of lipophorin it dropped to very low levels after 1 day. Lipophorin was shown to keep juvenile hormone III in solution and to reduce degradation of juvenile hormone III by corpora cardiaca-corpora allata. Thus, addition of lipophorin improves corpora cardiaca-corpora allata culture conditions and allows long-term culturing.

Strain-specificity of Galleria mellonella larvae to juvenilizing treatments

1. 1. The Warsaw and Florida strains of the wax moth, Galleria mellonella, are different in their sensitivity to juvenilizing treatments such as: chilling, application of ecdysone mimic (RH-5849) and juvenile hormone analogue (hydroprene), and implantation of brain or corpora cardiaca-corpora allata complex. 2. 2. The low sensitivity of the Florida strain larvae to these treatments results from low allatotropic activity of the brain of treated larvae and low sensitivity of corpora allata of this strain to allatotropin released from the brain. 3. 3. In the case of the Florida strain, the insects tissues had lower sensitivity to hydroprene and RH-5849 treatments compared to the Warsaw strain. 4. 4. In the haemolymph of chilled Warsaw strain larvae a small ecdysteroid peak, reaching a maximum of 28.0 ± 8.1 pg of 20-hydroecdysone per 1 μ1 of haemolymph was observed. 5. 5. This peak appeared 6 hr after chilling; it was absent in the haemolymph of chilled Florida strain larvae. 6. 6. The significance of this ecdysteroid peak in the induction of supernumerary larval moulting is discussed .

A monoclonal antibody to a 56 kDa neurosecretory polypeptide from the posterior lateral neurosecretory cells of the housefly, Musca domestica

A protocol was developed for generating monoclonal antibodies (MABs) to neurosecretory cells (NSCs) of the housefly, Musca domestica, using head extract as immunogen. A panel of MABs to housefly NSCs was produced using a 3-tier screening involving enzyme-linked immunosorbent assay (ELISA), western blot, and immunocytochemistry. One clone designated 01C10 was shown to immunostain the posterior lateral NSCs of the adult housefly brain. Immunostaining of neurohemal organs, corpora cardiaca (CC), was restricted to the late pharate adult stage and the first 6 h of adult eclosion. Loss of immunostained material from CC indicated its release into the hemolymph. Western blot studies revealed that 01C10 MAB immunoreacted with a 56 kDa polypeptide in the brain, corpora cardiaca/ corpora allata (CC/CA) complex of newly emerged adults and hemolymph collected 1 h after adult eclosion. These results strongly suggested the neurohormonal nature of the 01C10-immunoreactive polypeptide. We speculated the 56 kDa polypeptide to be bursicon based on a HPLC separation of hemolymph proteins from newly emerged adults which showed the co-elution of bursicon activity and the 56 kDa polypeptide in the same fractions.

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.

Isolation and identification of a new diuretic peptide from the tobacco hornworm, Manduca sexta

A 30-amino acid diuretic peptide was isolated from the corpora cardiaca-corpora allata complexes and, separately, from medial neurosecretory cells of the Sphingid moth, Manduca sexta. The peptide was found to have the following sequence, determined by automated Edman degradation and mass spectrometry: SFSVNPAVDILQHRYMEKV AQNNRNFLNRV-NH 2. We have named the peptide Mas-DP II. The peptide was synthesized and shown to possess diuretic activity in decapitated moths. Mas-DP II is related by sequence homology to a 41-amino acid diuretic peptide identified previously from M. sexta, and it belongs to the family of corticotropin releasing factor-like peptides.

The role of the frontal ganglion and corpora cardiaca/corpora allata complex in post‐feeding weight loss in adult Heliothis zea

Abstract A post‐feeding diuretic response has been observed in adult Heliothis zea (Boddie) (Lepidoptera: Noctuidae). 2‐day‐old starved adults which were allowed to feed to repletion on a 10% (w/v) sucrose solution lost 49.1% (females) and 85.8% (males) of the weight of the ingested meal during the first hour following feeding. Ligation between head and thorax or frontal ganglionectomy, when performed immediately following feeding, each resulted in a significant and permanent reduction in this normal weight‐loss. Injection of homogenates of the corpora cardiaca/corpora allata (CC/CA) complex into non‐ligated insects immediately after feeding also reduced significantly the post‐feeding weight‐loss, but this inhibition was transient and disappeared after 1 h. Dissection and weighing of the crop from either ligated, frontal ganglionectomized, or CC/CA‐injected insects confirmed the crop as the predominant site of fluid retention in each case. We suggest that a soluble antidiuretic factor from the CC/CA acts in conjunction with the frontal ganglion to control the rate of crop emptying and subsequent diuresis by regulating the volume of ingested fluid that is passed into the haemolymph from the crop/midgut.

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor—allatinhibin (AI)—from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 μl medium. CA must be exposed to AI for 12–16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.

Gastrin-cholecystokinin-like and neuroparsin-like immunoreactivities in the brain and retrocerebral neuroendocrine complex of the cockroach Blattella germanica

Using single and double labelling techniques respectively, brain-corpora cardiaca-corpora allata complexes of the cockroach Blattella germanica have been immunohistochemically investigated with antisera raised against either the vertebrate peptide gastrin-cholecystokinin (CCK-8(s] and/or the locust neurohormone neuroparsin (NPA). Single immunolabelling with anti-CCK-8(s) reveals immunoreactive perikarya and processes in median and lateral parts of protocerebrum, optic lobes, deutocerebrum and tritocerebrum. Some fibres originating in median and lateral protocerebrum are intrinsic to the brain, whereas others terminate in the nervous areas of the corpora cardiaca. Single immunolabelling with anti-NPA reveals immunoreactive cell bodies in the median part of the protocerebrum and their processes terminate both in the nervous area of the corpora cardiaca and between the intrinsic secretory cells of this neurohaemal organ. Double immunolabelling with anti-CCK-8(s) and anti-NPA enables a description of the anatomical relations between the processes and the endings of these two neurosecretory systems.

Immunohistochemical localisation of the hypertrehalosaemic hormone II (Cam-HrTH-II) and related peptides in the nervous system of Carausius morosus and Sarcophaga bullata.

A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the Lom-AKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.

Short-loop negative and positive feedback on ecdysone secretion by prothoracic gland in the tobacco hornworm, Manduca sexta

Ecdysteroid production by the prothoracic glands of the tobacco hornworm, Manduca sexta was found to be under feedback control by the ecdysteroids in hemolymph using both culture in vivo in diapausing pupae lacking the brain-corpora cardiaca-corpora allata complex and the prothoracic glands and culture in vitro. Prothoracic glands having relatively high activity in larvae, prepupae, or developing pupae were inhibited by ecdysone or 20-hydroxyecdysone. By contrast, prothoracic glands with low activity from feeding larvae, day 1 non-diapausing pupae and diapausing pupae were activated by both ecdysone and 20-hydroxyecdysone in vivo and in vitro. Dose-response studies on diapausing pupal glands showed that ecdysone was the most effective activator. These findings suggest that prothoracic glands are either stimulated or inhibited by ecdysone or 20-hydroxyecdysone, depending on both the secretory activity of the gland and the effective level of ecdysteroids in hemolymph. Thus, when the glands are first activated, the ecdysteroids that are secreted show a positive feedback on the glands to increase ecdysteroid output. Then the activated glands are turned off by the increasing 20-hydroxyecdysone titer in the hemolymph leading to the rapid decrease in ecdysteroid titer at the end of the molt period.

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

AbstractIn 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.

Hormonal Control of Adult Diapause in the Brown-Winged Green Bug, Plautia stali SCOTT (Heteroptera : Pentatomidae)

After the corpora allata (CA) were extirpated from non-diapausing adults of the brown-winged green bug, Plautia stali SCOTT, the adult colouration changed to reddish-brown and development of ovaries in females and ectodermal accessory gland (ectadenia) in males was inhibited. Implantation of corpora cardiaca-corpora allata complexes (CC-CA) into diapausing adults induced a green colouration and development of ovaries and ectadenia. Topical application of synthetic juvenile hormone (JH) III showed a similar effect to the implantation. These results indicate that JH has a crucial role in the hormonal control of adult diapause in P. stali. Further, the transection of nervous connections between the brain and CC-CA in diapausing adults revealed a diapause-terminating effect as did CC-CA implantation and JH III application. This suggests that JH-secretory activity is inhibited by the brain via nervous connections in diapausing adults.

Purification and characterization of eclosion hormone from the moth, Manduca sexta

The eclosion hormone (EH) of Manduca sexta is a 62 amino acid peptide which causes the behavioral and physiological changes that occur at ecdysis. EH was purified from the corpora cardiaca-corpora allata (CC-CA) complexes from pharate adult moths using acidic methanol extraction followed by two high performance liquid chromatography separation steps. One CC-CA complex was found to store about 3.5 ng of EH. Different techniques for estimating the molecular weight of the peptide, gel filtration chromatography and SDS-urea polyacrylamide electrophoresis, gave values of about 10 and 4 kD, respectively. Both were significantly different from the calculated molecular weight of 6813 dalton. The experimentally determined pI of the peptide was 4.8. Treatment of EH with various reagents showed that intact disulfide bridges are required for biological activity.

Stage-dependent biosynthesis of methyl farnesoate and juvenile hormone III and metabolism of juvenile hormone III in embryos of the cockroach, Nauphoeta cinerea

By culturing corpora cardiaca-corpora allata complexes from embryos in vitro in the presence of either [ 14C-methyl]methionine or [2- 14C]acetate we have shown that these glands synthesize methyl farnesoate and JH III de novo at stages after dorsal closure. Rates of production of these two substances were found to increase rapidly after dorsal closure and to remain on a similar level until a few days before hatching when a decrease was seen. Methyl farnesoate was by far the predominant product formed under in vitro as well as in vivo conditions until the stage of breaking of the chorion, whereafter JH III became predominant. Our in vitro analyses on JH III degradation in embryos revealed that the egg-case envelope efficiently degraded JH III at all stages investigated, whereas embryo homogenates degraded JH III efficiently only at stages shortly before hatching after the chorion had broken up. The major metabolite of embryos was identified as JH III acid and separate analyses of various tissues indicated that esterases were mainly located in fat body and/or integument and to some extent in gut and haemolymph.

Allatotropic activity of the median neurosecretory cells ofGalleria mellonella (lepidoptera) larval brain

The brain allatotropic hormone (ATTH) is released inGalleria mellonella from the Median neurosecretory cells located in the pars intercerebralis. These cells show the ability to elicit supernumerary larval molts upon implantation into sensitive host larvae, and the ability to in vitro stimulate the juvenile hormone synthesis in corpora cardiaca-corpora allata glands ofG. mellonella.

Interendocrine control by 20-hydroxyecdysone of the corpora allata of Manduca sexta

A positive interendocrine control by 20-hydroxyecdysone regulates the corpora allata (CA) and thus the hemolymph titers of the juvenile hormones (JHs) during the fourth and fifth larval stadia of the tobacco hornworm, Manduca sexta. The effect of 20-hydroxyecdysone is exerted via the brain-corpora cardiaca (Br-CC), and the kinetics of stimulation by the hormone suggest that its control of the CA is indirect, possibly involving neural effectors. Co-incubation of precommitment CA with Br-CC resulted in a stimulation of the synthesis of JH I acid equivalent to that achieved by the incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) with 20-hydroxyedcysone, suggesting that a diffusible regulatory factor may be involved in the stimulation. During pharate pupal development, a close temporal relationship exists between the drop in the ecdysteroid titer and declines in CA activity and the JH hemolymph titer, suggesting that 20-hydroxyecdysone may now inhbit the CA. Incubations of day 6 Br-CC-CA with physiological concentrations of 20-hydroxyecdysone resulted in an apparent dose-dependent inhibition of CA activity. Thus in Manduca, 20-hydroxyecdysone may exert varying stage-specific interendocrine effects on the CA which direct the postembryonic development of this species.

Juvenile hormones and their titer regulation in Galleria mellonella

Juvenile hormones I, II and III occur in Galleria but JH II is dominant. Its concentration reaches a peak of 3 pmol/g body wt in the penultimate instar, drops to zero in the last larval instar and, except for a small peak in prepupae (0.2 pmol/g), remains undetectable until pharate adults. After emergence the titer reaches over 5 pmol/g in both sexes. Presence of JH II is associated with JH II acid; JH III acid occurs even more often, including stages lacking JH III. Brain implantation into freshly ecdysed last instar larvae effects a similar JH peak as in the penultimate instar and causes an extra larval molt. The opposite treatment, i.e. decerebration of fresh last instar larvae, elicits a continuous rise of JH II to 10 pmol/g and an increase of otherwise rare JH I to 3 pmol/g. Sham operations of these larvae or decerebration of old larvae elevate practically only JH II titer to 1–1.5 pmol/g. Implanted brain-corpora cardiaca-corpora allata complexes maintain in various hosts 0.14–1.6 pmol/g of JH II. The significance and regulation of these fluctuations in JH titer are discussed.

Juvenile hormone degradation in brain and corpora cardiaca-corpora allata complex during the last larval instar ofGalleria mellonela (Lepidoptera, Pyralidae)

Time course analysis of juvenile hormone degradation in the brain and the corpora cardiaca-corpora allata complex shows that during the first two days of the last larval instar the juvenile hormone degradation is very low. Starting from the third day up to the seventh day a continuous increase of esterase activity is observed.