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.     

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.     

Quantitative studies on the release of locust adipokinetic hormone

Fractionation of methanolic extracts of haemolymph on Sephadex LH-20 made possible the measurement of the titre of adipokinetic hormone in the haemolymph of locusts. Experimentally produced high concentrations of haemolymph carbohydrate caused a delay in the mobilization of lipid during flight, and very low titres of the hormone were present in the haemolymph of locusts injected with trehalose immediately before a 25 min flight. In these locusts flight speed was higher than saline-injected controls. Although delayed lipid mobilization during flight was also seen in locusts injected with sucrose, sucrose is not utilized for flight metabolism and flight speed was not increased by the injection. Tentative estimates of the release rate (c. 1000pg/20min flight) and half life (c. 20 min) of adipokinetic hormone during flight are made. The results described suggest that during flight the rate at which trehalose disappears from the haemolymph does not play a major role in the initiation of the release of adipokinetic hormone.     

Cyclic AMP in locust fat body: Correlation with octopamine and adipokinetic hormones during flight

The effects of flight upon the level of cyclic AMP in the fat body of Locusta migratoria have been examined. Flight induced two phases of cyclic AMP elevation; the first during the initial 10 min of flight, the second between 20–30 min of flight. Neck-ligated locusts have increased levels of cyclic AMP after 10 min of flight, indicating that the adipokinetic hormones are not necessary for this elevation. Injection of 6 mg trehalose, a procedure known to delay the release of adipokinetic hormones, prevented the increases seen at 10 and 30 min of flight. Injection of synthetic adipokinetic hormone I increased the levels of cyclic AMP within 5 min, and these were maintained for up to 15 min. The roles of octopamine and the adipokinetic hormones in increasing fat body cyclic AMP, and thereby regulating haemolymph lipid, during flight are discussed.     

The hormonal control of haemolymph lipid during flight in Locusta migratoria

Fractionation of methanolic extracts of haemolymph on thin layer chromatography, followed by bioassay, has been used to measure the titres of adipokinetic hormones I and II in the haemolymph of flown locusts. These titres have been correlated with the elevation in haemolymph lipid. Haemolymph lipid elevates in a biphasic manner during locust flight. A rise in lipid occurs during the first 10 min of flight. Lipid levels then plateau between 10 and 20 min. A second, more pronounced elevation begins at 20 min and continues for up to 60 min. The titre of adipokinetic hormone I elevates 10–15 min after flight commences while that of hormone II elevates between 15–30 min. Adipokinetic hormone I contributes 80% of the activity at 30 min but only 45% at 60 min. It is suggested that the elevation in haemolymph lipid during the first 10 min of flight may not be induced by adipokinetic hormone I or II. The role of octopamine in this initial elevation is proposed and discussed.     

Release of identified adipokinetic hormones during flight and following neural stimulation in Locusta migratoria

Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci II (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the R_F value similar to Carlsen's peptide (adipokinetic hormone II).These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta, and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in ligated locusts.It is concluded that NCC II controls the release of adipokinetic hormones during flight and that two adipokinetic hormones are released during flight. One of these hormones adipokinetic hormone II also acts as a hyperglycaemic hormone illustrating that a hyperglycaemic hormone is released, during flight.     

Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed.     

The role of adipokinetic hormone in the control of vitellogenesis in locusts

Vitellogenesis in locusts is synchronized with the cyclic maturation of oocytes. Vitellogenesis by excised fat body of gravid females is differentially inhibited 80–90% when locust adipokinetic hormone I (AKH-I) is added to the incubation media. Hemolymph methanolic extracts completely inhibit fat body protein synthesis in vitro when the donor females are at the end of the ovarian cycle (6 mm stage), but not when taken from earlier stages. Hemolymph methanolic extracts from vitellogenic females at the 6 mm stage are separated by HPLC into three distinct inhibitors of protein synthesis, one of which is AKH-I. AKH-RIA of hemolymph during the first ovarian cycle reveals no AKH-I during active vitellogenesis, but a marked increase to about 5 ng per female at the end of egg maturation. A development of responsiveness to AKH-I is evident in female fat body as vitellogenesis proceeds. AKH-I is involved in the negative control of vitellogenesis.     

Induced hyperlipaemia and immune challenge in locusts

Injections of immunogens, such as beta-1,3-glucan or lipopolysaccharide (LPS), bring about a marked hyperlipaemia with associated changes in lipophorins and apolipophorin-III in the haemolymph of Locusta migratoria. These changes are similar to those observed after injection of adipokinetic hormone (AKH). The possibility that endogenous AKH is released as part of the response to these immunogens is investigated using passive immunisation against AKH-I, and measurement of AKH-I titre in the haemolymph after injection of immunogens. The data presented show that, despite the similarity of the changes brought about by the presence of immunogens in the haemolymph to those brought about by AKH, there is no release of endogenous AKH after injection of laminarin or LPS. A direct effect of the immunogens on release of neutral lipids by the fat body cannot be demonstrated in vitro, and the mechanism by which hyperlipaemia is induced during immune challenge remains uncertain.     

长管蚜激脂激素提取物生物活性的研究

激脂激素和高血糖激素已在某些昆虫种类中发现,但是到目前为止还未涉及到同翅目昆虫。本研究中,从同翅目昆虫长管蚜Mcrosiphum sp.体内分离了激脂激素提取物,并进行了生物活性测定。结果证明,这种提取物具有很高的生物活性,注射20毫克蚜虫重的提取物,能使试虫沙漠蝗与美洲蜚蠊体液中脂肪含量和糖含量分别增加131.03%和60.43%。 试验结果还展示出长管蚜激脂激素提取物激脂效应要比高血糖效应高得多,它们的生物活性能被内肽酶24∶11,焦谷氨酰胺基肽酶和沸水浴抑制或破坏。     

Physiological actions by hypertrehalosemic hormone and adipokinetic peptides in adult Blaberus discoidalis cockroaches

Members of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family characteristically cause metabolite mobilization by the insect fat body. The present study identified several additional physiological actions in adult Blaberus discoidalis cockroaches that were influenced by synthetic Blaberus hypertrehalosemic hormone (HTH) and other AKH/RPCH family peptides. HTH elevated blood carbohydrate by 4-fold and cytochrome heme a + b synthesis of fat body mitochondria by 3-fold. Both carbohydrate and heme synthesis were dose-responsive to HTH. Carbohydrate synthesis was 10 times more sensitive to HTH than heme synthesis. Heme synthesis was also stimulated by Periplaneta cardioacceleratory hormones (CAH)-I and -II and RPCH but not by AKH-I or -II, at the doses tested. HTH showed strong cardioexcitatory activity. Long-term treatment of decapitated female B. discoidalis with juvenile hormone analog (JHA = methoprene) stimulated by 2.6-fold the rate of synthesis of secreted fat body proteins. HTH enhanced the JHA-dependent export protein synthesis by 42% above that observed with JHA alone. SDS-PAGE demonstrated that JHA determined the nature of the newly synthesized polypeptides; HTH enhanced their synthesis rate. Neither AKH-I nor HTH affected protein synthesis when added directly to isolated fat body. These results demonstrate that peptides of the AKH/RPCH family have multiple physiological actions related to fat body energy metabolism.     

Eclosion hormone activity in haemolymph of eclosing silkmoth, Bombyx mori

The presence of eclosion hormone in the haemolymph of eclosing adults of the silkworm, Bombyx mori, was demonstrated to provide evidence that the hormone is involved in the induction of eclosion of this insect.The eclosion of the adult occurred within 55–75 min after light-on, when the pupae had been kept under conditions of 16 h light and 8 h dark during adult development. The hormone became detectable in the haemolymph 10–20 min after light-on, followed by eclosion 40 min later. The hormonal activity increased sharply to a maximal level (30 units/ml haemolymph) at 30 min prior to the eclosion and then fell rapidly: detectable eclosion hormone was present in the haemolymph for only 15–20 min. The hormone was partially purified from the haemolymph of eclosing animals and a 400-fold purification was achieved. The preparation was rather stable against heat treatment, and the molecular weight was estimated to be 5,000–10,000 by gel-filtration on a Sephadex G-50 (superfine). The origin of eclosion hormone in the haemolymph is discussed.     

Qualitative and quantitative changes in Locusta haemolymph proteins and lipoproteins during ageing and adipokinetic hormone action

Changes in haemolymph proteins and lipoproteins during adipokinetic hormone action have been studied using polyacrylamide gel electrophoresis (PAGE) and a heparin/EDTA precipitation technique. During hormone action, the formation of A⁺ takes place at the expense of Ayellow and C_L-proteins, which decrease in free concentration in the haemolymph. Ayellow is heparin precipitable, whereas A⁺ precipitates with EDTA after prior treatment with heparin. After injection of adipokinetic hormone, heparin-precipitable protein (HPP) decreases after a delay of 10–15 min, but heparin/EDTA precipitable protein (HEPP) increases immediately. These changes occur in response to extracts of corpora cardiaca and to synthetic adipokinetic hormone, and are dose-dependent. Both the lipid and the C_L-protein content of the HEPP rise as its protein content increases. A⁺ formation does not occur in fifth-instar nymphs or newly emerged adults, but this response to adipokinetic hormone develops slowly as the adults mature.     

Determination of locust AKH-I by radioimmunoassay and the identification of an AKH-like factor in the locust brain

The N-terminal glutamic acid analog of the decapeptide locust adipokinetic hormone-I, [Glu¹]AKH-I, was prepared by solid-phase synthesis and derivatized to [4-hydroxyphenyl propionyl-Glu¹]AKH-I. This derivative was radioiodinated on the 4-hydroxyphenyl function ([¹²⁵I]AKH analog). [Glu¹]AKH-I was coupled to bovine serum albumin to produce rabbit antiserum. This, together with the [¹²⁵I]AKH analog was utilized to develop a highly selective radioimmunoassay procedure (RIA) for detecting AKH-I in locusts. Methanol extracts of locust brain contain two factors (BI and BII) that inhibit protein synthesis in excised fat body tissue of vitellogenic female locusts. BI also elicits lipid mobilization when injected into adult male locusts. The HPLC retention time of BI on an RP-18 column is identical to that of locust adipokinetic hormone (AKH-I), both synthetic and derived from adult corpora cardiaca (CC). The second brain factor (BII) does not exhibit lipid mobilizing activity. One tissue-equivalent of BI contains 33 ng of AKH-I as determined by RIA, compared to 450 ng native HPLC-separated AKH-I in locust CC.     

Differences in adipokinetic response of Pyrrhocoris apterus (Heteroptera) in relation to wing dimorphism and diapause

Three experimental groups of adult females (reproductive and diapausing brachypters, and macropters with reproductive arrest) of Pyrrhocoris apterus (Linnaeus) (Heteroptera: Pyrrhocoridae) from a temperate population were analysed for their adipokinetic responses. The adipokinetic response, expressed as an increase of haemolymph lipids after injection of adipokinetic hormone from Locusta migratoria (Lom-AKH-I), was assessed in relation to age, wing dimorphism and type of reproductive arrest. Two pmols of Lom-AKH-I were used for determination of adipokinetic responses. The increase of haemolymph lipids in all experimental groups of females induced by this dose of the hormone was comparable with that induced by crude extract of the bug’s own corpora cardiaca. The level of adipokinetic response after injection of 2 pmol of Lom-AKH-I was significantly higher in macropterous and diapausing brachypterous females than in reproductive brachypterous females. However, significantly higher contents of haemolymph lipids in control macropterous females than those found in the control reproductive and diapausing brachypterous females of the corresponding age revealed wing-morph-related differences in lipid metabolism. The observed wing morph- and diapause-related differences in the content of haemolymph lipids and adipokinetic response, respectively, are discussed in relation to the different roles of two wing morphs in the life history of this heteropteran.     

Flight fuel related differences between solitary and gregarious locusts (Locusta migratoria migratorioides)

Abstract. Flight fuel relations of crowded and isolated Locusta migratoria migratorioides were investigated in younger (12–16 days after fledging) and older (27–30 or 27–32 days after fledging) adult males.No phase polymorphism dependent differences were found in resting haemolymph carbohydrate levels of the younger locusts.In the older age group, resting haemolymph carbohydrate levels were slightly though significantly higher in the isolated than in the crowded locusts.Injection of various doses of synthetic adipokinetic hormones (AKHs) did not induce marked changes in haemolymph carbohydrate levels and no differences were found between crowded and isolated locusts.A 30 min flight led to the same decrease in haemolymph carbohydrate levels of isolated and crowded locusts, 43.3% and 44.6% of the resting levels, respectively.We concluded, therefore, that the results do not seem to indicate that isolated locusts rely more heavily on carbohydrates as flight fuel than crowded locusts.Hyperlipaemic responses to flight were less intense in isolated than in crowded locusts, but phase polymorphism dependent differences in flight-induced increase of haemolymph lipid levels were not parallel in 12–16-day-old and 27–32-day-old males.In the younger age group the difference was mainly in the duration of flight needed to induce full response which appeared already after 20 min of flight in the crowded locusts, but only after 45 or 60 min of flight in the isolated ones.In contrast, the older isolated locusts showed markedly lower haemolymph lipid elevations than the crowded locusts even after 30, 45 or 60 min of flight.The hypothesis is forwarded that isolated locusts have a rather coarse adipokinetic strategy focused on a single long-distance migratory flight, whereas gregarious locusts possess a fine adipokinetic balance for reiterative migratory flights and saving fuel reserves for unpredictable long-distance migrations.     

Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body

We have previously shown that stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn -1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body. After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP. Forskolin (0.1 micrometer), an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids. Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body. Our findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers.     

Topical application of Pya-AKH stimulates lipid mobilization and locomotion in the flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)

Abstract Two different methods of applying Pya -AKH to long-winged (macropterous) females of the firebug, Pyrrhocoris apterus (Linnaeus) (Heteroptera) were compared: both injection and topical application increased the levels of lipids in the haemolymph and stimulated locomotor activity. Lipid mobilization was maximal when 10 pmol was applied by injection or 40–100 pmol by topical application, with the first significant responses occurring 1.5 h after injection and 2 h after topical application. The highest elevations of lipid concentration in the haemolymph were comparable between the treatments, i.e. 14.36 ± 3.59 mg/mL for injection and 14.43 ± 4.07 mg/mL for topical application. However, these maximal elevations were achieved at different times: 3 h after the injection and 7 h after the topical application.
Injection of 10 and 40 pmol of Pya -AKH stimulated locomotor activity with maximal activity 3 h later but, surprisingly, injection of 80 pmol showed no effect initially and than a slight inhibitory effect after 6–8 h.
Increased locomotor activity was found after topical application of Pya -AKH, but the response was lower than after injection and appeared later, 5–9 h after the hormone application. In addition, the greatest increase in walking activity required topical application of 300 pmol and was still less dramatic than the response to injection. The stimulatory effect of Pya -AKH on locomotion was positively correlated with its effect on lipid mobilization only for injection of the hormone. It is argued that a stress caused by injection could play a role in the appearance of the complex response to adipokinetic hormone.     

Substrate usage and its regulation during flight and swimming in the backswimmer, Notonecta glauca

Abstract.  The metabolites that are generally used by insects during exercise are present in quite different concentrations in the haemolymph of the backswimmer Notonecta glauca L. Lipids are most abundant (between 10 and 20 mg/mL), whereas carbohydrates (2–3 mg/mL) and proline (approximately 1 mg/mL) are at very low concentrations. Injection of an extract of conspecific corpora cardiaca causes pronounced hyperlipaemia in the backswimmer. A neuropeptide with the same effect was isolated from the corpora cardiaca in a single high-performance liquid chromatography (HPLC) step; the primary sequence was deduced from mass spectrometric measurements (matrix-assisted laser desorption/ionization-time of flight and electrospray quadrupol time-of-flight mass spectrometry) of whole corpora cardiaca, and the mass was confirmed in the HPLC fraction that had adipokinetic activity. The biologically active octapeptide has the sequence pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide, which was characterized previously from the corpora cardiaca of the Emperor dragonfly, Anax imperator , and denoted Anaim-adipokinetic hormone (AKH). The synthetic Anaim-AKH peptide causes lipid mobilization when injected at a dose of 1 pmol into N. glauca . When other synthetic AKH members that occur in Hemiptera are injected into N. glauca at the same dose, the hyperlipaemic responses are significantly lower than after injection of Anaim-AKH. Because only lipids increase upon activity, such as continuous swimming for 1 h or during a 1-h rest period after a 3-min flight episode in the laboratory, it is assumed that Anaim-AKH serves as a true adipokinetic hormone in the backswimmer during bouts of natural swimming and flight.     

CL-proteins and the regulation of lipoprotein lipase activity in locust flight muscle

Lipoprotein lipases in the flight muscles of Locusta migratoria show a marked substrate specificity: diacylglycerols associated with the adipokinetic hormone (AKH)-induced lipoprotein, A+, are hydrolysed at 4 to 5 times the rate of those associated with the lipoprotein in resting (non-hormone-stimulated) locusts, Ayellow. To determine the basis for this discrimination, the effect on the activity of flight muscle lipoprotein lipase of CL-proteins, a major constituent of lipoprotein A+, but not of Ayellow, has been investigated; they inhibit the flight muscle enzyme in a competitive manner whether activity is measured with a natural lipoprotein substrate, a lipid emulsion or a water soluble substrate. Experiments in vivo suggest that the flight muscle enzyme is normally inhibited in resting (non-AKH-stimulated) locusts but, interestingly, injection of synthetic AKH-I relieves the inhibition and increases the activity by 30 to 40%. This is not a direct effect of the hormone on the enzyme, but appears to be related to the hormone-induced formation of lipoprotein A+, so that the majority of CL-proteins in the haemolymph become bound to this lipoprotein and the concentration of free CL-proteins is markedly reduced. We suggest that CL-proteins play a major role in the regulation of lipoprotein lipase in locust flight muscle.