Adipokinetic hormone content of the corpora cardiaca in gregarious and solitary migratory locusts

Abstract. The adipokinetic hormone (AKH-I and AKH-II) content of the corpora cardiaca from adult males of crowded (gregarious) and isolated (solitary) Locusta migratoria migratorioides (Reiche & Fairmaire) was quantified by reverse-phase high-performance liquid chromatography.Significantly less total hormone was found in the corpora cardiaca of crowded locusts than in those glands of isolated locusts at the age of 12–19 days after fledging.The ratio of AKH-I/AKH-II was higher in crowded than in isolated locusts at this age.From the age of 12–19 days to that of 25–30 days, AKH content increased significantly in the corpora cardiaca of crowded locusts, but no such increase was found in the glands of isolated locusts, and at 25–30 days there were no significant differences in the AKH content of the glands from crowded and isolated locusts.     

Rapid in vitro activation of corpora allata by extracted locust brain allatotropic factor

Brains of young (newly emerged) adult female locusts (Locusta migratoria migratorioides) and of mature (> 9 days old) locusts contain an extractable allatotropic factor, soluble in 100% methanol and in distilled water. This factor stimulates juvenile hormone III (JH III) synthesis and release from corpora allata (CA) that have been excised from donor locusts and then incubated with (radiolabeled methyl)-methionine in vitro in its presence. In addition to JH III, which is the major product synthesized by the CA, other hexanesoluble, radiolabeled compounds–-more polar than JH III–-are also released when CA are incubated in vitro. The activation of CA by the allatotropic factor is rapid and quickly declines when the factor is removed from the medium. Corpora allata excised from young females are marginally active and can be activated by brain allatotropic factor to less of an extent than CA of mature locusts. The content of allatotropic factor in brains of mature locusts is higher than that ascertained in brains of young females. Allatotropic factor is also present in the corpora cardiaca.     

Substrate utilization and flight speed during tethered flight in the locust

Mature laboratory locusts normally exhibit a characteristic pattern of change in flight speed with time. They fly at high speed for the first few minutes, during which carbohydrate forms the major fuel, but then slow to a cruising speed when lipid is used almost exclusively. Locusts flown for 30 min, rested for 2hr, and then reflown, exhibit an identical pattern of flight, even though they oxidise only half the amount of carbohydrate used in the first flight. The injection of adipokinetic hormone before the first flight elicits a low initial flight speed for 10 to 15 min but then the locusts accelerate to a constant higher speed. The injection of hormone before the second flight, when blood lipid levels are already high, reduces the utilization of carbohydrate by the flight muscles dramatically but results in constant high-speed flight.     

The inhibition of lipid synthesis in vitro in the locust, Schistocerca gregaria, by factors from the corpora cardiaca

ABSTRACT. The rate of lipid synthesis from [¹⁴C]acetate in fat body from Schistocerca americana gregaria has been studied in vitro. Maximum incorporation is found on days 6–10 in adults and day 4 of the fifth stadium. The label appeared in the fatty acid components of triacyl-glycerol, diacylglycerol and phospholipid.
Lipid synthesis in vitro was inhibited by extracts of corpora cardiaca, and such inhibition was most marked (up to 85%) in fat bodies from insects at stages where fatty acid synthesis was greatest. HPLC separation of corpora cardiaca extracts gave several active fractions of which the most active was adipokinetic hormone 1 (AKH-1).     

Preliminary characterization of glycogen phosphorylase activating hormone and adipokinetic hormone from Manduca sexta corpora cardiaca

ABSTRACT. An attempt was made to separate glycogen phosphorylase activating hormone (GPAH) and adipokinetic hormone (AKH) from the corpora cardiaca (CC) of the moth Manduca sexta (Lepidoptera: Sphingidae) by separating extracts of CC on various chromotographic media, but it was not possible to conclude whether GPAH and AKH are activities of one or of two different peptides. Both activities elute together from glass beads, from Sephadex G-25 and from Sephadex LH-20 columns. In the separation experiments with glass beads and G-25 the activities eluted as a single peak, but using LH-20 we found two peaks exhibiting both activities. The major peak eluted at 1.25 × V_t, which is very similar to locust AKH, while the smaller second peak eluted at O.74 × V _t. Cross injections of CC extracts from M. sexta into Locusta migratoria and CC extracts from L. migratoria into M. sexta suggest that GPAH and the AKH from M. sexta are not identical with the decapeptide AKH from locusts.     

Further characteristics of adipokinetic and hyperglycaemic factor(s) of stick insects

When an extract of the corpora cardiaca/corpora allata from two species of wingless stick insects, Carausius morosus and Cuniculina impigra, which cause no adipokinetic or hyperglycaemic effect when injected into the donor insects themselves, is injected into adult Locusta migratoria it resulted in an increase in the haemolymph lipid concentration. The lipid elevation was time dependent, with a maximum effect about 90–180 min after injection, and was also dose-dependent. About 0.001–0.002 (C. morosus) and 0.01 (C. impigra) gland equivalents were needed to produce a significant increase; a maximal effect was reached with approx. 0.075 (C. morosus) and 0.25 (C. impigra) gland equivalents. Carausius extract was also able to elevate carbohydrate concentration in the haemolymph of Periplaneta americana. However, the effect was weak and no maximal response was reached even with a dose of 0.5 gland equivalents. Adipokinetic hormone activity was present in CC/CA extracts of larval Carausius; the activity was about 30 times lower in 1-day-old 2nd instar individuals, and approx. 5 times less at the beginning of the 6th instar than that found in adults. In both stages the hormone levels increased gradually from the beginning to the end of the instar. No age-related changes were observed during the adult stage. Further studies on the lipid-mobilising factor of C. morosus revealed that it was stored entirely in the CC and not in other nervous tissue, e.g. brain, CA, suboesophageal ganglion, thoracic and abdominal cord. The factor was heat stable for at least 1 hr at 100°C and retained its adipokinetic activity after incubation with trypsin and the exopeptidases such as carboxypeptidase A and leucine aminopeptidase. However, activity was abolished when incubated with thermolysin and α-chymotrypsin. From these experiments a close resemblance to the locust AKH, a blocked decapeptide, is suggested.     

The dark-colour-inducing neurohormone of locusts in relation to an albino mutant of Schistocerca gregaria

In the albino mutant of an Okinawa strain of Locusta migratoria (L.) (Orthoptera: Acrididae), albinism is caused by the absence of the dark‐colour‐inducing neurohormone (DCIN), which is present in the corpora cardiaca (CC) of normally coloured phenotypes. This study tests whether the absence of DCIN is responsible for albinism in an albino mutant of another locust, Schistocerca gregaria (Forsk.) (Orthoptera: Acrididae). This seemed feasible because a single Mendelian unit controls albinism in both species. However, implantation of CC, or injection of an extract of CC, from albino donors of S. gregaria, induce dark coloration in crowded nymph recipients of the Okinawa albino mutant of L. migratoria, as effectively as do implanted CC, or injections of extract of CC, from normal phenotype donors of S. gregaria. Therefore, DCIN is present in the albino mutant of S. gregaria, and consequently, the albinism in this mutant is not caused by its absence. Implantation of CC, or injection of extracts of CC, from albino donors of S. gregaria to conspecific albino nymphs does not induce darkening. Only extremely high doses of synthetic DCIN injected into albino nymphs of S. gregaria are effective, inducing some darkening. The dose to induce such darkening in albino nymphs of S. gregaria is 50 nmol, ≈ 5 × 10⁶ times higher than that (10 femtomol) needed to induce equivalent darkening in nymphs of the Okinawa albinos of L. migratoria. The results are discussed and some possible explanations of the observed effects outlined.     

An investigation into the effects of stimulators of fluid production on Locusta Malpighian tubule intracellular elemental composition

The intracellular elemental concentrations of Na, K, P, S, Cl and Mg in the type 1 cells of Malpighian tubules of Locusta migratoria L. have been measured using electron probe X-ray microanalysis. The effects of in vitro stimulation with 1 mM cAMP and corpora cardiaca extract (CC-extract) on the elemental concentrations have been quantified. The distribution of elements, particularly Na, K and Cl is not homogeneous in control cells, and concentration gradients exist within the cytoplasm. Dibutyryl-cAMP (DB-cAMP) caused a decrease in [K]_i without disrupting the gradient which increased from the basal to the apical surface, the apical [Na]_i was increased as was the [Cl]_i. In contrast, in vitro application of CC-extract did not cause changes to the intracellular elemental composition as compared with control cells These data are consistent with the interpretation that exogenous cAMP only partially activated the full stimulatory response of Malpighian tubule cells observed with CC-extract. The changes observed in the density and elemental composition of the `dark bodies' in response to DB-cAMP and CC-extract stimulation suggest that these structures have a role in the ionic economy of Malpighian tubule cells. Accepted: 6 April 1999     

Immunochemical analysis of the distribution of the new ovary maturating neurohormone during development of the African locust,Locusta migratoria

Summary Following our prior identification of a gonadotropic neurohormone isolated from the neurosecretory lobe of the corpora cardiaca of the African locust, we have raised a polyclonal antiserum against this new molecule. In the present paper, we characterize this antiserum using enzyme-linked immunosorbent assay and Western blotting. The latter procedure reveals that the immune serum specifically recognizes the neurohormone, which we have termed ovary maturating parsin. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to analyze the distribution of this gonadotropic neurohormone throughout the central nervous system during development. It is produced only by the type-B neurosecretory cells of the pars intercerebralis-corpora cardiaca system and is present both in males and females throughout life from embryo to adult. This permanent expression suggests that the neurohormone may have functions other than its primary direct gonadotropic role in females.     

Variability in Juvenile Hormone production by locust corpora allata kept in vitro for long periods

Abstract. Juvenile Hormone III (JH-III) production by corpora allata (CA) of sexually mature female locusts (Locusta migratoria migratorioides (R. & F.)) was maintained in vitro for up to 30 days in an agar-solidified medium. Hormone production was measured periodically with a short-term radiochemical assay. Low-activity CA increased their activity significantly after 24–48 h incubation in the long-term medium, but high-activity glands did not. Variations in activity were considerable among glands tested on the same day and among measurements from the same gland on different days. Farnesoic acid-stimulated rates of JH-III production were always higher than the basal rates, suggesting that the CA were not maximally activated. However, freshly excised low-activity CA, whose hormone production increased in the long-term conditions, showed similar farnesoic acid-stimulated rates of JH-III production to those of freshly excised high-activity glands, suggesting that at the time of excision of the corpora allata rate-limiting step(s) preceding farnesoic acid biosynthesis were inhibited or refractory to stimulation in vivo.     

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.     

Lipoprotein transformations during adipokinetic hormone action in Locusta migratoria

ABSTRACT. Detailed studies of lipoprotein A⁺ formation during AKH action have been made in Locusta migratoria L. using gel filtration combined with the use of radiolabeled haemolymph protein probes. In addition, we have assessed the quantitative contribution of the C_L-proteins to A⁺ formation by direct measurement of the changes in concentration of free C_L-proteins and report some properties of the C-I and C-II proteins: they appear to be glycoproteins of 20,000 and 16,000 MW respectively, but do not bind to concanavalin A. We have confirmed earlier observations (using different techniques) which showed that liproprotein A_yellow is not involved per se in A⁺ formation during the first 15 min of AKH action. In contrast, the (two) C_L-proteins take part in A⁺ formation without any apparent delay after hormone injection. Our observations show that A⁺ formation is essentially complete within 30 min of AKH injection, although further C_L-protein binding and lipid-loading do occur subsequently. After 30 min there is no further decrease in the A_yellow titre. It is argued that much, if not all, C_L-protein is located at the surface of the A⁺ particle. From the changes in titres which occur in A_yellow and C_L-proteins during AKH action we estimate that A⁺ is formed from 1 mole of A_yellow and approximately 28 moles of C_L-proteins. Using these figures we calculate an apparent molecular weight for A⁺ within the range of 1.65–2.12×10⁶, which is in reasonable agreement with estimates derived from gel exclusion chromatography data. These studies emphasize the dynamic and fully reversible nature of lipoprotein A⁺ formation and highlight the complex nature of the lipoprotein transformations occurring during hormone-stimulated lipid transport in locusts.     

Osmotic lysis of corpora cardiaca using distilled water reveals the presence of partially processed peptides and peptide fragments

Abstract. A simple, single-step aqueous extraction method has been developed to study the neuropeptide content of small neuroendocrine organs. Perifusion of these tissues with deionized water causes osmotic bursting of the cells and release of their content into the surrounding fluid. The neuropeptides are immediately retained from the perifusion fluid using disposable C18 cartridges. After one separation step and mass spectrometry, it was possible to identify a large number of known neuropeptides from the corpora cardiaca of Locusta migratoria (L). Also present in the extract were a number of neuropeptide fragments and two incompletely processed peptides. Using this method, a 959Da peptide present in the corpora cardiaca was sequenced de novo . The full sequence, deduced using Collision Induced Dissociation Tandem Mass Spectrometry (CID MS/MS), is Ser-Pro-Leu-Asp-Ala-His-His-Leu-Ala. This nonapeptide is predicted from the gene encoding the ion transport peptide precursor and from the gene encoding the ion transport-like peptide precursor. In both cases, this nonapeptide, which was named ion transport peptide-copeptide, is flanked by the signal sequence at the N -terminus and a dibasic cleavage site (Lys-Arg) at the C -terminus. This structural feature is common to many physiologically important locust preproneuropeptides and indicates that this copeptide might have a physiological function, but this is not yet known.     

Relationship of adipokinetic hormone I and II to migratory propensity in the grasshopper, Melanoplus sanguinipes

This report examines three aspects of adipokinetic hormone (AKH) involvement in migratory flight behavior in the grasshopper, Melanoplus sanguinipes. The titer of hemolymph AKH I during long-duration tethered flight was examined using radioimmunoassay (RIA) after narrow bore RP-HPLC. The hemolymph fraction containing AKH I was assayed using commercially available anti-Tyr1-AKH I serum. Titer determinations of hemolymph AKH were done at rest and after various periods of flight. The amount of AKH I released from the corpora cardiaca during flight was estimated. When resting levels of AKH I and II in corpora cardiaca (CC) of migrants and non-migrants were examined with HPLC, no significant differences in AKH levels were detected between non-migrants, animals that had flown for 1 h to identify them as migrants, and animals that had flown to exhaustion (i.e., voluntary cessation). CC levels of both AKH I and II were less in this species than in locusts. When the lipid mobilization in response to AKH I and II was compared in migrants (animals that had self-identified as migrants in a 1-h tethered flight test) and non-migrants (animals that would not perform a 1-h flight in a tethered flight test), the adipokinetic response to AKH I was greater in migrants than in non-migrants, possibly indicating differences in level of sensitivity or number of receptors in the target tissues. AKH II had little effect on hemolymph lipid levels in either flight group, and may not play a significant role in lipid mobilization in this species.     

Hormonal control of lipid synthesis in the fat body of the adult female tsetse fly, Glossina morsitans

Aqueous extracts of brain, thoracic ganglion or corpora cardiaca of female Glossina morsitans were shown to contain a substance which inhibited the synthesis of lipid from l[U-¹⁴C] leucine by fat cells incubated in vitro. The highest concentration of this substance was found in the corpora cardiaca; approximately 1 × 10^?6 gland pairs μl^?1 were required for maximum inhibition. At concentrations greater than 1 × 10^?4 gland pairs μl^?1 the lipid synthesis inhibiting factor (hereafter referred to as the LSIF) was inactivated by the presence of a substance which could be removed by gel filtration. The concentration of LSIF in the corpora cardiaca and midbrain varied throughout the reproductive cycle of the female. Net release of LSIF from the midbrain occurred between the 2nd and 7th day of the 9-day reproductive cycle. Net release from the corpora cardiaca began on day 5 and continued until the end of the interlarval period on day 9. Results are consistent with the hypothesis that LSIF is synthesised mainly in the medial neurosecretory cells of the midbrain whereas the corpora cardiaca are the site of storage and release into the haemolymph. LSIF was present in midbrain and corpora cardiaca extracts from male G. morsitans but at lower concentrations than in females. No variation in LSIF concentration could be correlated with the feeding cycle. LSIF activity was not detected in fresh haemolymph but was found at high concentration in boiled haemolymph, suggesting the presence of an inhibitor which was inactivated at high temperature. Preliminary investigations into the nature of LSIF have shown it to be inactivated by proteolytic enzymes and to be recoverable in a single peak from a Sephadex G15 column.Results support the view that LSIF is a peptide hormone which, in conjunction with an inhibitor, controls the lipid synthetic ability of the fat cells of the adult female tsetse fly throughout the reproductive cycle.     

Identification of gonadotropic and antigonadotropic factors from the nervous part of the corpora cardiaca in the African locust

Summary

The nervous part of the locust corpora cardiaca (NCC) was extracted using 70% methanol and subjected to fast protein liquid chromatography (FPLC). Twenty fractions were collected and injected into females from the end of the IIIrd larval instar to day-12 of the adult life to screen the effects on metamorphosis, body pigmentation, and oocyte growth. The rate of juvenile hormone (JH) biosynthesis by the corpora allata of injected animals was evaluated in vitro. One fraction was found to stimulate ovarian maturation, and 3 to delay oocyte growth. None of them disturbed (i) either metamorphosis and body color (which are JH dependent), or (ii) rate of JH biosynthesis. Of the 3 fractions which inhibited ovarian maturation, 2 of them were identified as the neuroparsins A and B. The results clearly illustrate that antagonist factors from the brain are involved in the regulation of ovarian maturation in the locust.     

The effect of constant darkness on the content of adipokinetic hormone, adipokinetic response and walking activity in macropterous females of Pyrrhocoris apterus (L.)

Abstract.  Changes in the content of adipokinetic hormone (AKH), the adipokinetic response and the walking activity of 10-day-old adult macropterous females of the firebug, Pyrrhocoris apterus (L.), reared under long-day (LD) photoperiod (LD 18 : 6 h) are compared with those exposed for 3 days to constant darkness (DD). Diel changes of all the parameters studied in LD females persist in females kept in constant dark. A positive correlation exists between diel changes of AKH content in the central nervous system (CNS) in the LD and DD females, and a negative correlation in the AKH level in haemolymph and walking activity. In addition, there is a positive correlation between diel changes of AKH level in haemolymph and walking activity in macropterous females reared under LD conditions, as well as in those transferred to constant darkness. The data suggest that there is some feedback between the release of AKH from CNS into the haemolymph and walking activity in macropterous females. Preliminary studies on the simultaneous expression of mRNA for the period gene and a positive reaction to an antibody against AKH in the same corpus cardiacum cells suggest that the period gene may be involved in regulating the AKH content in this gland.     

Adipokinetic hormone of the true armyworm, Pseudaletia unipuncta: immunohistochemistry, amino acid analysis, quantification and bioassay

Abstract A cluster of five to seven AKH-like immunoreactive cells lie in each lobe of the paired corpora cardiaca of the true armyworm, Pseudaletia unipuncta. These cells form a mesh work of immunoreactive processes within the corpora cardiaca, and immunoreactive tracts projecting posteriorly over the aorta.
Reversed-phase high performance liquid chromatography of extracts of the corpora cardiaca of P.unipuncta revealed a single large U.V. absorbent peak with a retention time identical to synthetic Manduca- AKH. Amino acid analysis of the contents of this peak yielded a composition identical to that of synthetic Manduca-AKH which was analysed in a parallel manner. Furthermore the material within the peak possessed adipokinetic activity when bioassayed in day 2 adult male P. unipuncta. The corpora cardiaca of similar individuals were found to contain approximately 17.6ng (17.6pmol) of Manduca-AKH equivalents per pair.
Injection of Manduca-AKH into 2-day-old adult male P.unipuncta resulted in a dose-dependent elevation in haemolymph lipid levels with a maximum level of 80–90μmg/μl obtained with 5–10 ng of Manduca-AKH. Continuous flight also elevated haemolymph lipid levels in day 4 adult males with a significant elevation evident in the first samples taken after 15 min of flight and lipid levels plateauing at approximately 100 μg/μl by about 60 min of flight.     

Development of imaginal competence to adipokinetic hormone in Locusta: lipid and carbohydrate levels, and glycogen phosphorylase activity in precocene-induced adultiforms

Abstract. Adipokinetic responses to injection of synthetic adipokinetic hormone I (sAKH) were investigated in precocene-induced fifth-instar adultiforms and in chemically allatectomized but morphogenetically normal adults of Locusta migratoria (L.). The results were compared with data on normal fifth (=last)-instar nymphs and normal adults. Chemical allatectomy did not affect the resting level of lipid in the haemolymph, nor the slight decrease of haemolymph carbohydrate concentration induced by sAKH. The effects of chemical allatectomy on sAKH-induced hyperlipaemia, the resting level of haemolymph carbohydrate, total glycogen phosphorylase specific activity, as well as sAKH-induced phosphorylase activation in the fat body, were mostly minor and probably indirect. The concentrations of lipid and carbohydrate in the haemolymph, and sAKH-induced activation of fat body glycogen phosphorylase were more or less similar in normal fifth-instar nymphs, normal adults and fifth-instar adultiforms. Thus, precocious metamorphosis did not affect markedly those parameters which show no or slight changes during normal metamorphosis. In contrast, parameters which change substantially during normal metamorphosis (sAKH-induced hyperlipaemia, sAKH-induced changes in haemolymph carbohydrate level and total glycogen phosphorylase activity) showed similar changes in the course of precocious metamorphosis; the values for fifth-instar adultiforms were similar to those obtained for normal adults, but differed markedly from those found in normal fifth-instar nymphs. Thus, accelerated (precocious) morphological adult development is strongly correlated with accelerated imaginal target competence to AKH and with relevant physiological development.