The effect of adipokinetic hormone on midgut characteristics in Pyrrhocoris apterus L. (Heteroptera).

Digestive processes and the effect of adipokinetic hormone (Pyrap-AKH) on the amount of nutrients (lipids, proteins, and carbohydrates), and on the activity of digestive enzymes (lipases, peptidases, and carbohydrases) were studied in the midgut of the firebug, Pyrrhocoris apterus. The analyses were performed on samples of anterior (AM), middle (MM) and posterior (PM) midgut parts. The results revealed that the digestion of lipids, carbohydrates and proteins take place in the acidic milieu. The Pyrap-AKH treatment increased significantly the level of lipids and proteins in the midgut, and also the level of triacylglycerols (TGs) predominantly in the AM, and the level of diacylglycerols (DGs) in the MM. The increase was not uniform for all present TG and DG species - those containing the linoleic fatty acid were predominant. No hormonal effect on lipase activity was recorded, while peptidase and glucosidase activity was increased in the MM and PM. All these facts indicate that the Pyrap-AKH probably stimulates digestion by more intensive food ingestion or turnover, and perhaps by the stimulation of metabolite absorption; the activation of digestive enzymes seems to be secondary or controlled by other mechanisms.

Oxidative stress elicited by insecticides: a role for the adipokinetic hormone.

Adipokinetic hormones (AKHs) are insect neuropeptides responding to stress situations including oxidative stress. Two insecticides - endosulfan and malathion - were used to elicit oxidative stress conditions in the firebug Pyrrhocoris apterus, and the physiological functions of AKHs and their ability to activate protective antioxidative reactions were studied. The insecticide treatments elicited only a slight increase of the AKH level in CNS, but more intensive increase in haemolymph, which indicates an immediate involvement of AKH in the stress response. The treatment also resulted in a significant increase of catalase activity in the bug's body and depletion of the reduced glutathione pool in the haemolymph, however, co-application of the insecticides with the AKH (80 pmol) reduced the effect. It has also been found that co-application of the insecticides with AKH increased significantly the bug mortality compared to that induced by the insecticides alone. This enhanced effect of the insecticides probably resulted from the stimulatory role of AKH on bug metabolism: the carbon dioxide production was increased significantly after the co-treatment by AKH with insecticides compared to insecticide treatment alone. It was hypothesized that the increased metabolic rate could intensify the insecticide action by an accelerated rate of exchange of metabolites accompanied by faster penetration of insecticides into tissues.

A novel function of red pigment-concentrating hormone in crustaceans: Porcellio scaber (Isopoda) as a model species.

The RP HPLC and LC/MS QTOF analyses of the methanolic CNS extract from isopod crustacean the woodlouse, Porcellio scaber revealed a presence of the red pigment-concentrating hormone (Panbo-RPCH) in this species. It has been shown that this neuropeptide plays a role in mobilization of energy stores: topical treatments of P. scaber individuals by Panbo-RPCH in a concentration 20 pmol/microl increased the level of glucose in haemolymph about 4 times, while the level of trehalose was only doubled. The results demonstrated that glucose was the main carbohydrate mobilized by the Panbo-RPCH treatment: glucose was responsible for about 97% of total carbohydrate increasing. Despite the demonstration of hyperglycaemic activity of Panbo-RPCH, no stimulatory effect of this hormone on the locomotory activity of P. scaber was observed. The present study is the first discovery of an occurrence of Panbo-RPCH and its hyperglycaemic activity in the representative of the isopod crustaceans. The relationship of the function of Panbo-RPCH in P. scaber to the role of this neuropeptide and adipokinetic hormones in insects is discussed.

Adipokinetic hormone (Pyrap-AKH) enhances the effect of a pyrethroid insecticide against the firebug Pyrrhocoris apterus.

BACKGROUND: Adipokinetic hormones (AKHs) are insect neuropetides controlling stress situations including those elicited by insecticide treatment. The effect of Pyrap-AKH on the mortality of the firebug Pyrrhocoris apterus (L.) treated with the insecticide permethrin (Ambush 25 EC) was studied. RESULTS: Coinjection of 50 ng permethrin with 80 pmol Pyrap-AKH induced a significant 2.3-fold increase in bug mortality compared with the insecticide alone. The results were confirmed by topical coapplication of both agents (400 ng and 80 pmol respectively). Injections of 50 and 100 ng permethrin elicited a significant increase in the AKH level in CNS and the haemolymph. The results indicate an involvement of AKH in stress response to permethrin. The enhanced effect of insecticide by AKH treatments probably results from the stimulatory role in bug metabolism: carbon dioxide production was increased 3.5- and 2.5-fold respectively 1 and 3 h after permethrin treatment, and 4.3- and 3.4-fold after the permethrin plus AKH cotreatment, compared with the control. CONCLUSION: The elevation of metabolism could intensify the permethrin action by its faster penetration into tissues and by stimulation of biochemically active cells, and could be a reason for enhanced action of permethrin after its cotreatment with Pyrap-AKH.

Activation of insect anti-oxidative mechanisms by mammalian glucagon.

Resembling the main function of insect adipokinetic hormones (AKHs), the vertebrate hormone glucagon mobilizes energy reserves and participates in the control of glucose level in the blood. Considering the similarities, the effect of porcine glucagon was evaluated in an insect model species, the firebug Pyrrhocoris apterus. Using the mouse anti-glucagon antibody, presence of immunoreactive material was demonstrated for the first time in the firebug CNS and gut by ELISA. Mammalian (porcine) glucagon injected into the adult bugs showed no effect on hemolymph lipid level or on the level of AKH in CNS and hemolymph, however, it activated an antioxidant response when oxidative stress was elicited by paraquat, a diquaternary derivative of 4, 4'-bipyridyl. Glucagon elicited the antioxidant response by increasing glutathione and decreasing protein carbonyl levels in hemolymph, decreasing both protein carbonyl and protein nitrotyrosine levels in CNS. Additionally, when co-injected with paraquat, glucagon partially eliminated oxidative stress markers elicited by this redox cycling agent and oxidative stressor. This indicates that glucagon might induce an antioxidant defense in insects, as recently described for AKH. Failure of glucagon to alter AKH level in the bug's body indicates employment of an independent pathway without involving the native AKH.

Stimulatory effects of bioamines norepinephrine and dopamine on locomotion of Pyrrhocoris apterus (L.): is the adipokinetic hormone involved?

In the present paper we studied the effects of five biogenic amines - norepinephrine, dopamine, octopamine, serotonin and histamine - on the locomotory activity and mobilization of lipids in the adult females of the firebug, Pyrrhocoris apterus (L.). We tested the hypothesis (1) whether the stimulation of walking activity in the bugs injected with the bioamines is associated also with their hyperlipaemic effects, like in the case of adipokinetic hormones (AKHs), and (2) whether these effects are direct or mediated through a release of the AKHs into the hemolymph. The results demonstrated that all five tested biogenic amines mobilized the fat body lipids, but only norepinephrine and dopamine were capable to enhance the walking activity simultaneously with an elevation of the lipid level in the hemolymph. Those two amines had no effect on the level of AKHs in CNS, but modulated the AKHs level in hemolymph: norepinephrine increased it, while dopamine decreased it. The results indicate an apparent feedback between AKH characteristics and dopamine and norepinephrine actions occurring in this insect species. While the stimulatory effects of norepinephrine on lipid mobilization and walking activity could involve the release of bug's own AKHs, dopamine probably employs an independent stimulatory pathway.

Adipokinetic hormone-induced enhancement of antioxidant capacity of Pyrrhocoris apterus hemolymph in response to oxidative stress.

The in vivo effects of oxidative stress on adipokinetic hormone (AKH) titer in short-winged (brachypterous) males of the firebug Pyrrhocoris apterus were tested using paraquat (PQ), a bipyridilium herbicide. PQ undergoes a cyclic redox reaction with oxygen during microsomal and electron transfer reactions forming free radicals in the insect body. Oxidative insult (40 pmol PQ) resulted in enhanced protein carbonylation (a biomarker for oxidative stress) and a depletion of glutathione (GSH) pool in the hemolymph. Interestingly, AKH titer was significantly enhanced in hemolymph at 4 h post inoculation of PQ, while its content in CNS (brain with corpora cardiaca) showed non-specific changes in comparable period. Co-injection of AKH with PQ (40 pmol each) reversed these effects by decreasing protein carbonyl formation, increasing reduced GSH levels, and enhancing the total antioxidant capacity of cell free plasma. Our results indicate that there is a positive feedback regulation between an oxidative stressor action and the level of AKH in insect body, and that AKHs might be involved in the activation of antioxidant protection mechanism.

Flight muscles polymorphism in a flightless bug, Pyrrhocoris apterus (L.): developmental pattern, biochemical profile and endocrine control.

The flightless bug Pyrrhocoris apterus (L.) is polymorphic for both wing length and flight muscle development. The developed flight muscles of macropterous adults of both sexes first enlarge their volume during the first 5 days after adult emergence, but are then histolyzed in all males and females older than 10 and 14 days, respectively. The flight muscles of brachypterous adult males and females are underdeveloped due to their arrested growth. The total protein content of histolyzed dorsolongitudinal flight muscles from 21-day-old macropterous adults of both sexes is lower than that of developed dorsolongitudinal flight muscles in 5-10-days-old macropterous bugs, but substantially higher than the protein content of underdeveloped dorsolongitudinal flight muscles from adult brachypters. Histolyzed dorsolongitudinal flight muscles differ from the developed ones by decreased quantities of 18 electrophoretically separated proteins. Histolysis of developed dorsolongitudinal flight muscles is accompanied by significant decreases in citrate synthase, glyceraldehyde-3-phosphate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase enzyme activities and an increase in alanine aminotransferase activity, and can be precociously induced by application of a juvenile hormone analogue. This is the first report of flight muscle polymorphism, histolysis of developed flight muscles and its endocrine control in insects displaying non-functional wing polymorphism.

Wing morph-specific differences in the metabolism and endocrine control of reserve mobilization in adult males of a flightless bug, Pyrrhocoris apterus (L.) (Heteroptera).

The differences in the metabolism and endocrine control of reserve mobilization in long-winged (macropterous) and short-winged (brachypterous) males of a flightless firebug (Pyrrhocoris apterus) were studied. We found that protein content in the gut was significantly lower in 5-10 day-old macropterous males due to their fasting and higher in 28 day-old ones than in the same aged brachypterous counterparts as the result of renewed food intake. Overall protease activity was significantly lower in 10-14 day-old macropters, while an abrupt increase in the activity starting on day 21 after adult ecdysis was also associated with renewal of the food intake. The levels of carbohydrates in haemolymph were only slightly lower in 1-10 day-old macropterous males than in the same aged brachypters. However, more than twofold higher lipid content in haemolymph of 7-10 day-old macropterous males than in the same aged brachypterous males was found. Higher mobilization of lipid reserves from the fat bodies in macropterous males was accompanied by more intensive adipokinetic response and higher levels of adipokinetic hormone in the body. It is the first report of endocrine regulation of wing morph-related differences in the lipid mobilization in males of wing-polymorphic insects.

The effect of insecticide on adipokinetic hormone titre in the insect body.

The effect of an insecticide, permethrin (Ambush 25 EC), on the titre of two adipokinetic hormones in the central nervous system (CNS) and haemolymph of the firebug Pyrrhocoris apterus (L) was tested. Doses of 2.5, 12.5 and 25 ng of the insecticide elicited no significant effect on the titre of the adipokinetic peptides in CNS, but caused their dramatic elevation in the haemolymph of both macropterous and reproductive brachypterous bugs. Changes in diapausing brachypterous bugs were minimal and not significant. Using high-performance liquid chromatography (HPLC) and competitive ELISA, both adipokinetic peptides of P apterus (Pyrap-AKH and Peram-CAH-II) were identified and quantified in the CNS of all experimental groups. The ratios of the two peptides in the CNS of the bugs used were affected by insecticide treatment, but in all three groups of bugs the Pyrap-AKH level remained dominant. The insecticide-treated bugs were compared with those exposed to forced running (shaking) and dark shock. Both factors also acted as stressors and enhanced the titre of adipokinetic peptides in the haemolymph but had no effect on hormone titre in CNS. The results indicate an involvement of adipokinetic peptides in the response of insect to various stressors including insecticides.

Wing morph-related differences in developmental pattern of accessory gland proteins in adult males of Pyrrhocoris apterus (L.) and their endocrine control.

The study showed that the amounts of the total proteins and 53 kDa protein in male accessory glands (AGs) of the firebug Pyrrhocoris apterus (L.) increased with age of the adult life. The 53 kDa protein, the most abundant polypeptide detected in the secretion of the AGs, and some other smaller peptides were identified as glycoproteins. Changes in the amounts of the total proteins and 53 kDa protein in AGs were found to be wing morph-dependent and their levels were significantly higher in 1-10 days old brachypterous males than in macropterous males of the same age. Macropterous males were characterized by delayed growth of the AGs. Treatment of adult macropterous males with methoprene significantly increased the amounts of total proteins and 53 kDa protein in their AGs when compared to acetone-treated macropterous controls of the same age. Allatectomy of brachypterous males decreased the levels of the total proteins and 53 kDa protein in their AGs, while application of methoprene enhanced the quantity of allatectomy-suppressed proteins in these tissues. This is the first report of juvenile hormone-dependent wing morph-related differences in the synthesis of AG proteins and their endocrine control in wing-polymorphic insects.

Identification of the cockroach neuropeptide Pea-CAH-II as a second adipokinetic hormone in the firebug Pyrrhocoris apterus.

A new member of the AKH/RPCH family was isolated from the corpora cardiaca of the firebug Pyrrhocoris apterus. It is the second adipokinetic peptide identified in this species. The peptide was characterized and its structure was deduced from the multiple MS(N) electrospray mass spectra as that of an octapeptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH(2.) The peptide differs from the original P. apterus AKH (Pya-AKH) by one amino acid in position 3. Topical application and/or injection of the peptide induced lipid mobilization, but was inactive in mobilization of carbohydrates.