Barry G. Loughton

Octopamine and short-term hyperlipaemia in the locust.

Abstract The response of locusts to experimental handling has been examined. “Excitation” induced by handling resulted in an elevation of both hemolymph octopamine and hemolymph lipid levels. Octopamine levels rose within 2 min, while protein-bound lipid rose within 5 min. Both responses followed a similar time course with the levels falling between 1 and 2 hr following excitation. Lipid levels did not return at this time in neck-ligated locusts but remained high. The excitation induced hyperlipaemia was reduced by injection of the α-adrenergic blocking agent phenoxybenzamine. Injection of octopamine potentiated hyperlipaemia in neck-ligated locusts. The possibility of a cause-and-effect relationship between octopamine levels and lipid levels is discussed.

In vitro studies on the effects of octopamine on locust fat body.

Abstract The effects of octopamine on the fat body of locusts in vitro have been examined. The isolated fat body responded to octopamine by releasing lipid in a dose-dependent manner. Maximal stimulation was obtained with 5 × 10−6 M octopamine. The octopamine stimulated release of lipid was blocked by α-adrenergic receptor antagonists but unaffected by β-adrenergic receptor antagonists. Octopamine was more potent than synephrine, dopamine, or tyramine in eliciting the response. Octopamine (5 × 10−6 M) in the presence of IBMX, resulted in a six- to sevenfold elevation of cyclic AMP in the fat body; this response was also blocked by α- but not β-adrenergic receptor antagonists. Cyclic GMP levels were unaffected by octopamine. Adipokinetic hormone (AKH) in the presence of IBMX, also stimulated an elevation in cyclic AMP in the fat body, and stimulated the release of lipid. Unlike the octopamine stimulation, however, the AKH effect was not inhibited by the α-adrenergic receptor antagonists. Furthermore, AKH inhibited protein synthetic activity of fat body, while octopamine was without effect. It is concluded that a release of lipid from fat body of locusts is probably mediated by a rapid activation of an octopamine-sensitive adenylate cyclase. Furthermore, the mechanism of action and the receptors for octopamine and AKH are different.

An investigation of haemolymph protein economy during the fifth instar of Locusta migratoria migratorioides

Abstract Tritium-labelled haemolymph protein and [3H]-leucine were used to study haemolymph protein economy during the fifth instar of Locusta. Blood volumes and total haemolymph protein levels were also determined. Haemolymph volume increased by a factor of 3 and total haemolymph protein increased by a factor of 4 as the instar progressed. The periods of protein uptake were detected. One in the middle of the instar and a second more important one during the larval-adult ecdysis. The specific activities of soluble tissue proteins from insects injected with [3H]-blood-protein were high at the beginning of the instar and then gradually fell to a more or less constant level. Over 80 per cent of the [3H]-leucine injected was absorbed from the haemolymph throughout the instar. Incorporation of [3H]-leucine into haemolymph protein was highest near the midpoint of the instar. Specific activities of the soluble protein from midgut and flight muscle of locusts injected with [3H]-leucine was high at the beginning of the instar then fell to a steady level. The specific activity of fat body soluble protein was highest 3 days after ecdysis. The results are discussed in terms of protein synthesis, uptake, and turnover during the fifth instar.

An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust

Abstract The fate of haemolymph proteins during the fifth instar of Locusta has been investigated autoradiographically using tritiated blood protein. The fate of [ 3 H]-leucine was also studied. All tissues took up labelled blood protein at all stages of development. Pericardial cells were the most active tissue in protein uptake throughout the instar. The amount of label in oenocytes, fat body, and epidermis was high at the beginning of the instar, decreased after 136 hr, then rose once more after 200 hr. Low levels of activity were detected in the cuticle at the beginning of the instar. The new cuticle was evident after 200 hr and showed extensive labelling. A different pattern of labelling was evident after injection of labelled leucine. Once again label was evident in all tissues. The labelling was heavier than that observed in blood protein injected animals. During the first 100 hr of the instar, most tissues were heavily labelled. From 100 to 150 hr the level of uptake decreased. There was a second rise in incorporation of amino acid between 150 and 160 hr. The level of incorporation then decreased once more and remained low until the end of the instar. The new cuticle was the only tissue heavily labelled at the end of the instar. The significance of these results and the possible methods of protein uptake are discussed.

An oviposition-stimulating factor in the male accessory reproductive gland of the locust, Locusta migratoria

The data derived from mating experiments demonstrate that mating has an accelerating effect on oviposition in the female Locusta migratoria. The effect of mating could be mimicked by injection of extracts of the male accessory reproductive gland. The oviposition-stimulating factor was localized in the opalescent gland of the male accessory gland and was transferred to the female via the spermatophore during copulation. Gel filtration of an extract of the opalescent gland revealed a 13,000 Da protein, which, when injected into virgin female locusts, could stimulate the oviposition rate to that seen in mated females. Extracts of the corpus cardiacum also stimulated oviposition when injected into virgin female locusts. This increase was not observably different from that seen after mating. The relevance of these findings will be discussed.

The inhibition of protein synthesis in Locusta migratoria by adipokinetic hormone

Abstract Injection of glandular lobe extracts or synthetic adipokinetic hormone caused a suppression of haemolymph protein synthesis in adults in a dose-dependent manner. The inhibition of protein synthesis occurred at lower doses of adipokinetic hormone than that necessary to cause mobilization of lipid. The effect lasted approximately the same time as the elevation of haemolymph lipid. At higher doses, the hormone was less effective in inhibiting protein synthesis, though lipid mobilization did not change. Incubation of isolated half fat bodies in vitro in the presence or absence of either glandular lobes or adipokinetic hormone confirmed the in vivo results. Protein synthesis in heart and midgut tissue was also inhibited by adipokinetic hormone. Both arylphorin and vitellogenin synthesis were inhibited upon injection of the hormone into vitellogenic female locusts.

Neural inhibition of egg-laying in the locust, Locusta migratoria

Abstract The role of the oviducal nerves during egg-laying in Locusta migratoria has been examined. Section of the oviducal nerves did not inhibit egg-laying in any observable way. Electrical stimulation of the oviducal nerves resulted in a contraction of the common and lower lateral oviducts which propelled ovulated eggs up towards the ovaries. Recordings from oviducal nerves using chronically implanted electrodes showed that electrical activity was low during actual egg-laying, but high at times when egg-laying was not occurring (i.e. during digging behaviour, or following interruption of egg-laying). During these periods of high activity recurrent bursts of action potentials occurred. Similar patterns of electrical activity were recorded in semi-intact preparations using suction electrodes applied to exposed oviducal nerves of locusts which had been interrupted during the process of egg-laying. High frequency bursts of activity were recorded simultaneously from both left and right oviducal nerves. It is concluded that one function of the oviducal nerves is to inhibit egg-laying at inappropriate times, by inducing contractions of the oviducts which propel eggs back towards the ovaries. These nerves therefore provide a physiological basis for part of the adaptive ovipositional activities of locusts.

The nature of the hyperglycaemic factor from the glandular lobe of the corpus cardiacum of Locusta migratoria

Abstract Extracts of the glandular lobe of the corpus cardiacum increased haemolymph carbohydrate levels in neck-ligated adults of Locusta migratoria . No change was observed in normal locusts. Synthetic AKH had no effect on haemolymph carbohydrate in neck-ligated insects. Fractionation of the glandular lobe extract by gel filtration yielded a fraction which possessed both hyperglycaemic and hyperlipaemic activity in neck-ligated insects. Crustacean red-pigment-concentrating hormone possessed hyperglycaemic activity. It is surmised that the ‘second peptide’ (AKH II) functions, at least in part, to regulate blood sugar levels.

Tissue specific transduction systems for octopamine in the locust (Locusta migratoria)

Abstract The treatment of locust abdominal fat body in vitro with octopamine caused an elevation in cyclic 3′, 5′-adenosine monophosphate (cAMP). However the values varied from one tissue sample to another. Incubation of air sacs separated from fat body cells with octopamine reduced this variability and revealed that octopamine caused a massive increase in air sac cAMP levels but did not alter fat body cAMP concentrations. The air sac cells responded to octopamine in a dose-dependent fashion with a maximum response (10−5 m ) 40 fold above control levels. The increase in air sac cAMP was sufficient to account for all the increase observed when air sacs and fat body together were incubated with octopamine. Treatment of air sacs with forskolin at 10−5 m caused an increase in cAMP but no response was observed with fat body. Octopamine and forskolin acted synergistically to raise air sac cAMP concentrations. The data suggest that air sacs have the potential to make an active response to physiological demands placed upon the insect. The nature of that response is not known. In addition, the absence of changes in cAMP concentration in isolated fat body indicates that cAMP is not the second messenger for octopamine stimulated lipid release from fat body cells. This argues that the octopamine receptor on fat body cells has not yet been characterized.

Characterization of proctolin binding sites on locust oviduct membranes

Abstract The binding of [ 3 H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K d of 400 ± 82 nM and a B max of 23.7 ± 6.7 pmol/mg protein. The second had a K d of 2.4 ± 0.2 μ M and a B max of 96.3 ± 16.7 pmo/mg protein. Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [ 3 ]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.