Some problems of homeostasis of haemolymph metabolites in the locust

Sectioning of the afferent nerves (NCCI and II) to the locust corpora cardiaca, glandular lobe removal, cardiacectomy, or removal of the median neurosecretory cells of the brain, have no long-term effect on blood lipid concentration. After removal of the glandular lobe, haemolymph carbohydrate concentration is lowered and remains significantly so from the second to the sixth day after the operation but returns to normal within 10 to 15 days. Severance of the afferent nerves to the corpora cardiaca does not, however, affect blood carbohydrate concentration. The injection of a concentrated extract of glandular lobes into locusts deprived of their glandular lobes does not elicit a hyperglycaemic effect even when blood carbohydrate levels are low. Cauterization of the median neurosecretory cells of the brain, sectioning of the NCCI and II, or removal of the glandular lobes of the corpora cardiaca have no effect on haemolymph protein.After dilution of haemolymph constituents by the injection of water, carbohydrate and protein concentrations are not rapidly restored to their initial values. The lipid concentration, however, rapidly returns to its pre-injection level due to the mobilization of 16:16, 16:18, and 18:18 diglycerides. This occurs even in glandular lobe deprived, median neurosecretory cell cauterized, or headless locusts. These diglycerides are mobilized following the injection of solutions containing lipid, carbohydrate, and/or protein, and are the same diglycerides that are released from the fat body in response to adipokinetic hormone. It is concluded that the injection of large volumes of fluid causes lipid mobilization but adipokinetic hormone does not apear to be involved, and the mechanism of blood lipid homeostasis in the resting locust is not clear.     

Restricted specificity of a hyperglycaemic factor from the corpus cardiacum of the stick insect Carausius morosus

Extracts of the corpora cardiaca of the stick insect Carausius morosus elevate the haemolymph carbohydrate concentration in adult and 6th-instar larvae which are ligated behind the first pair of legs, but not in non-ligated (intact) insects. The increase in haemolymph sugars is due to trehalose elevation, is time dependent (with a maximal effect about 90–120 min after injection), and is dose dependent (needing 0.005 gland equivalents for a significant effect and a tenfold higher dose for a maximal response). The hyperglycaemic factor is localised entirely in the corpora cardiaca and appears to be specific to stick insects; corpora cardiaca extracts of two lepidopteran species (Acherontia atropos and Aglais urticae) and of Locusta migratoria have no effect, whereas corpora cardiaca extracts of the stick insects Cuniculina impigra and Sipyloidea sipylus have similar activity to those from C. morosus. This specificity is also shown when S. sipylus is used as the recipient. Synthetic adipokinetic hormone and red pigment concentrating hormone possess no hyperglycaemic activity in the stick-insect system. Two peaks of hyperglycaemic activity were obtained after column chromatography of corpora cardiaca extract of C. morosus on Sephadex G-25 and Sephadex LH-20. The factor seems to act via activation of fat-body glycogen phosphorylase, which, although 60% active in the control insects, is significantly increased to approx. 85% upon corpora cardiaca injection. However, the activation is demonstrated in ligated and intact insects. No significant decrease in the glycogen level of the fat body is observed after corpora cardiaca injection.     

The role of the glandular lobes of the corpora cardiaca during flight in Locusta

ABSTRACT. Flight performance in Locusta is reduced following severance of the major afferent nerves to the corpora cardiaca or removal of the glandular lobes of the corpora cardiaca. These operations prevent the release of adipokinetic hormone and the consequent mobilization of stored lipid. However, locusts deprived of about 90% of their glandular lobe tissue, while flying poorly, did mobilize lipid. It is suggested that the remaining glandular parenchyma cells are capable of secreting enough hormone to stimulate lipid mobilization, but that the concentration may be inadequate to encourage lipid utilization. After removal of all the glandular lobe parenchyma, the blood carbohydrate concentration was temporarily depressed. Nevertheless flight performance was equally poor, both when haemolymph carbohydrate levels were low and when they had returned to normal. After the injection of trehalose into operated control locusts and locusts deprived of their glandular lobes, flight was still markedly poorer in the operated insects, even though the injection of trehalose prevented adipokinetic hormone release in the intact locust. It seems that the poor flight performance of locusts deprived of their glandular lobes cannot be fully explained by the simple absence of adipokinetic hormone.     

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 effect of Metarhizium anisopliae var acridum on haemolymph energy reserves and flight capability in the desert locust, Schistocerca gregaria

Adult desert locusts, Schistocerca gregaria , 3 days after inoculation with the entomopathogenic fungus Metarhizium anisopliae var acridum , had significantly less carbohydrate and lipid in the haemolymph than controls. This was not due to reduced food intake as 3 days of complete starvation had no effect on haemolymph titres of energy reserves in controls. Furthermore injection of an extract of the corpora cardiaca (the source of adipokinetic hormone, AKH) caused a large significant increase in haemolymph lipid in mycosed locusts, indicating the availability of significant quantities of lipid in the fat body, the target for AKH. Haemolymph carbohydrate declined significantly during tethered flight of control locusts but not in mycosed individuals. An injected supplement of trehalose significantly boosted flight performance of mycosed insects but not controls. The results are discussed in the light of the hypothesis that the poor flight capability of mycosed locusts is due in part to a fungus-induced reduction in mobile energy reserves.     

Mobilization of lipid and carbohydrate reserves in the migratory grasshopper Melanoplus sanguinipes

Abstract. The North American migratory grasshopper Melanoplus sanguinipes Fabricius (Orthoptera: Acrididae) exhibits heritable variation in predisposition to make long-duration flights, and performance of long-duration flight enhances reproductive output. As a first step in understanding the physiological basis of these phenomena, we examined the mobilization of lipid and carbohydrate reserves during flight and in response to injection of extracts of the corpora cardiaca. Extract of conspecific corpora cardiaca elevates the concentration of haemolymph lipid. Both synthetic locust adipokinetic hormone I (AKH I) and synthetic Locusta migratoria AKH II raise the concentration of lipid in the haemolymph. However, although AKH I is more active than AKH II in locusts, dose-response curves for the two peptides are similar in M.sanguinipes. Neither extract of conspecific corpora cardiaca nor locust AKH I affects haemolymph carbohydrate in this species. Haemolymph carbohydrate and total glycogen reserves are Diminished by tethered flight; in contrast, haemolymph lipid is elevated by flight. Grasshoppers identified as presumptive migrants or non-migrants do not differ significantly in body composition. Total lipid reserves did not decrease measurably after extended flight, even though total reserves of carbohydrate do not appear to be sufficient to maintain the durations of flight performed.     

Role of methionine-enkephalin on the regulation of carbohydrate metabolism in the rice field crab Oziotelphusa senex senex

In the present study, the role of eyestalks and involvement of methionine-enkephalin in the regulation of haemolymph sugar level was studied. Bilateral eyestalk ablation significantly decreased the haemolymph sugar levels, whereas injection of eyestalk extract into ablated crabs significantly increased the haemolymph sugar levels. Total carbohydrate (TCHO) and glycogen levels were significantly increased in hepatopancreas and muscle of eyestalk-ablated crabs, with a decrease in phosphorylase activity. Injection of eyestalk extract into ablated crabs resulted in partial/complete reversal of these changes. Injection of methionine-enkephalin into intact crabs significantly increased the haemolymph sugar level in a dose-dependent manner. Total tissue carbohydrate and glycogen levels were significantly decreased, with an increase in phosphorylase activity in hepatopancreas and muscle tissues of intact crabs after methionine-enkephalin injection. Methionine-enkephalin injection did not cause any changes in haemolymph sugar, tissue total carbohydrate and glycogen levels and activity levels of phosphorylase in eyestalk-ablated crabs. These results suggest that the eyestalks are the main source of hyperglycaemic hormone and methionine-enkephalin induces hyperglycaemia through eyestalks.     

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.     

Adipokinetic hormone counteracts oxidative stress elicited in insects by hydrogen peroxide: in vivo and in vitro study

The role of adipokinetic hormone (AKH) in counteracting oxidative stress elicited in the insect body is studied in response to exogenously applied hydrogen peroxide, an important metabolite of oxidative processes. In vivo experiments reveal that the injection of hydrogen peroxide (8 µmol) into the haemocoel of the firebug, Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae) increases the level of AKH by 2.8‐fold in the central nervous system (CNS) and by 3.8‐fold in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental insects, whereas co‐injection of hydrogen peroxide with Pyrap‐AKH (40 pmol) reduces mortality to almost control levels. Importantly, an increase in haemolymph protein carbonyl levels (i.e. an oxidative stress biomarker) elicited by hydrogen peroxide is decreased by 3.6‐fold to control levels when hydrogen peroxide is co‐injected with Pyrap‐AKH. Similar results are obtained using in vitro experiments. Oxidative stress biomarkers such as malondialdehyde and protein carbonyls are significantly enhanced upon exposure of the isolated CNS to hydrogen peroxide in vitro, whereas co‐treatment of the CNS with hydrogen peroxide and Pyrap‐AKH reduces levels significantly. Moreover, a marked decrease in catalase activity compared with controls is recorded when the CNS is incubated with hydrogen peroxide. Incubation of the CNS with hydrogen peroxide and Pyrap‐AKH together curbs the negative effect on catalase activity. Taken together, the results of the present study provide strong support for the recently published data on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting oxidative stress. The in vitro experiments should facilitate research on the mode of action of AKH in relation to oxidative stress, and could help clarify the key pathways involved in this process.     

Haemagglutinin activity in the haemolymph of individual Acrididae (grasshopper) specimens

Twenty-four individual grasshopper specimens representing four Melanoplus spp. contained similar broad-spectrum haemolymphatic haemagglutinin. The agglutinin activity showed highest titre toward human ABO and rabbit cells among nine types of erythrocytes tested. Titre values differed between individual insects but agglutination specificity toward different erythrocytes was similar. Agglutination of type-O red cells by individual grasshopper haemolymph was inhibited by 34 of 41 tested carbohydrates, carbohydrate derivatives, alcohols and chelating agents. Individual insects showed similar patterns of haemagglutination inhibition. Non-inhibitory compounds were mannose and mannose derivatives (excepting N-acetylneuraminate), several glucose derivatives, amino sugars and ethanol. The observations indicated that haemolymph from an individual grasshopper contained complex heteroagglutinin activity similar to that found in haemolymph pooled from several insects. Determination of minimal effective inhibitor concentrations confirmed the presence of heteroagglutinin activity primarily directed toward galactose and glucose and related α-linked glycosidic derivatives.     

The adipokinetic hormones of African water bugs of the Heteropteran families Nepidae and Belostomatidae

Four African species of true water bugs (Nepomorpha: Hemiptera: Heteroptera) are studied by mass spectrometry and biological assays to gain information on the presence, structure and function of peptides from the adipokinetic hormone (AKH) family, which are produced in the corpora cardiaca (CC). The water scorpion Laccotrephes fabricii Stål (Nepidae) has the peptide code‐named Peram‐CAH‐I with the sequence pGlu‐Val‐Asn‐Phe‐Ser‐Pro‐Asn‐Trp amide, whereas Appasus grassei Poisson (Belostomatidae) produces Anaim‐AKH, which is a Ser⁷ analogue of Peram‐CAH‐I (pGlu‐Val‐Asn‐Phe‐Ser‐Pro‐Ser‐Trp amide). The giant water bug Hydrocyrius columbiae Spinola (Belostomatidae) has two adipokinetic hormone family members: Anaim‐AKH and Letin‐AKH, which again differ only at position 7 (Ser⁷ versus Tyr⁷). When the sequence data are compared with current molecular phylogenetic analyses of Nepomorpha, they are essentially in agreement with the newest ideas on phylogenetic relationships among the families. Functional investigation of these peptides reveals a mainly lipid‐based energy metabolism in these insects, as demonstrated by a hyperlipaemic response after injecting crude CC extract or the appropriate peptide into the respective species. The carbohydrate concentration in the haemolymph is not affected by such injections, and the carbohydrate level in most cases is lower than that of the circulating lipids. During physical exercise, such as swimming for 1 h, carbohydrates may contribute to some extent to the provision of energy; the substantial increase in the concentration of lipids in the haemolymph, however, is a strong indicator that the peptides are released from the CC and act primarily as true adipokinetic hormones during this period of intense muscular activity.     

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.     

Morbidity and mortality in Norway lobsters, Nephrops norvegicus: physiological, immunological and pathological effects of aerial exposure

A multivariate approach has been used to study progression in the post-capture condition of trawl-caught Nephrops norvegicus destined for the live transport market. A range of biochemical (L-lactate, glucose, glycogen), endocrinological (crustacean hyperglycaemic hormone), immunological (total haemocyte counts, phenoloxidase), microbiological and pathological measures of condition were utilised. During prolonged periods of aerial exposure N. norvegicus experience large disruptions to the carbohydrate profile, with increases in haemolymph L-lactate and crustacean hyperglycaemic hormone concentrations, and corresponding fluctuations in haemolymph pH; the severity of this disruption increases with the temperature of aerial exposure. This in turn impacts on the immune competence of the lobsters, with significant reductions in the number of circulating haemocytes and phenoloxidase levels observed as well as increases in the degree of bacteraemia of the haemolymph. Utilising evidence obtained during histological and other studies, possible causes of the immuno-suppression and subsequent meat spoilage are discussed. The information obtained should help to identify critical periods in the post-capture period that promote poor stock condition and mortality. Such data may be used to generate an internationally accepted Code of Practice for the capture, handling and transport of commercially exploited decapod crustaceans.     

Neuroparsin: A new energetic neurohormone in the African locust

The effects of neuroparsins on hemolymph trehalose and lipid levels and on total glycogen content were analyzed in Locusta migratoria. Saline and methanol extracts of the two lobes of the corpora cardiaca were assayed. Neuroparsins (A and B) were demonstrated to be hypertrehalosemic and hyperlipemic proteins of the neural lobe. Both of these metabolic activities of neuroparsins were somewhat less potent than those of adipokinetic hormone (AKH). Neuroparsin activity could be distinguished from AKH by blockage with an antiserum specific to neuroparsin. The hypertrehalosemic response induced by neuroparsins, in contrast to that of AKH, appeared to occur without a decrease of total glycogen content. The differential modes of action of AKH and neuroparsins could contribute to the fine modulation of carbohydrate metabolism in Locusta migratoria.     

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.     

Hyperglycaemic effects of 5-hydroxytryptamine and dopamine in the freshwater prawn, Macrobrachium malcolmsonii

The effects of 5-hydroxytryptamine (5-HT; serotonin) and dopamine (DA) on tissue carbohydrate metabolism and haemolymph glucose levels in the freshwater prawn, Macrobrachium malcolmsonii, were investigated. Injection of 5-HT and DA produced hyperglycaemia in a dose-dependent and time-dependent manner, with DA being more effective than 5-HT. Interestingly, 5-HT and DA induced hyperglycaemia only in intact prawns but not in bilaterally eyestalk-ablated individuals. Total carbohydrate (TCHO) and glycogen levels decreased and phosphorylase activity increased in the hepatopancreas and muscle of intact prawns after being injected with 5-HT or DA. However, bilateral eyestalk ablation decreased haemolymph glucose and tissue phosphorylase activity and increased TCHO and glycogen levels of the hepatopancreas and muscle. Injection of 5-HT or DA did not cause significant changes in these variables in eyestalk-ablated prawns. It is hypothesized that 5-HT and DA induce hyperglycaemia in prawns by stimulating the release of crustacean hyperglycaemic hormone (CHH) from the X-organ sinus gland (XO-SG) complex located in the eyestalk.     

A quantitative study of adipokinetic hormone of the firebug,Pyrrhocoris apterus

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/μl.     

Identification of the adipokinetic hormone of the large milkweed bug Oncopeltus fasciatus

Abstract The adipokinetic hormone (AKH) of the large milkweed bug Oncopeltus fasciatus is isolated from an acidified methanolic extract of 200 corpora cardiaca, purified by single step reversed phase high‐performance liquid chromatography (HPLC) and N‐terminally deblocked using pyroglutamate aminopeptidase. The sequence is identified by Edman degradation and matrix assisted laser desorption/ionization‐time of flight mass spectroscopy as pGlu‐Leu‐Asn‐Phe‐Ser‐Pro‐Asn‐Trp amide. This structure is confirmed by chemical synthesis and coelution of native and synthetic peptide on HPLC. The AKH of O. fasciatus is identical to Tenmo‐HrTH, a member of the adipokinetic/red pigment‐concentrating hormone peptide family that had been isolated earlier from several tenebrionid beetles. Tenmo‐HrTH causes a significant rise in the concentration of haemolymph lipids when injected into adult male and female O. fasciatus, but displays no hyperglycaemic activity. There is no indication of the presence of other AKHs in O. fasciatus. The large milkweed bug represents the first member of the seed bugs (Lygaeidae) for which the endogenous AKH has been identified.     

Lipid mobilization and locomotor stimulation in Gryllus bimaculatus by topically applied adipokinetic hormone

Abstract.  Walking activity of 3-day-old adult female Gryllus bimaculatus (de Geer) (Ensifera, Gryllidae) was measured over 24 h. A high level of locomotor activity during the scotophase was found, which was two- to three-fold higher than that during the photophase. The titre of lipid in the haemolymph was relatively low 2 h after lights on, increased significantly 2 h after lights off, although, 2 h after lights on in the next photophase, the lipid titre had decreased to the basal level. Topical application of homologous Grybi-adipokinetic hormone (AKH) (100 pmol in 20% 2-propanol) led to a significant increase in haemolymph lipids, comparable with the maximal increase caused by injection of AKH (3 pmol in water). Topical application of AKH also stimulated locomotor activity in crickets (maximal stimulation 1.8-fold with 100 pmol Grybi-AKH). The results suggest that AKH penetrates the cuticle quickly. It is assumed that AKH stimulates locomotory activity at least in part via the increase of haemolymph lipid titres; however, the stimulation of locomotor activity via a direct neuromodulatory effect of AKH cannot be excluded.