FMRFamide-like activity in the female locust during vitellogenesis.

The control of oviposition in the locust involves the expulsion of eggs from the lateral oviducts, a process believed to be under neurohormonal control. In this paper we have attempted to identify this putative hormone. Immunohistochemical staining of the brain retrocerebral complex and suboesophageal ganglion of Locusta migratoria with antiserum against FMRFamide revealed a number of FMRFamide-immunopositive cells. FMRFamide-like immunoreactivity was present in median neurosecretory cells and lateral neurosecretory cells of the protocerebrum. Other FMRFamide-immunoreactive cells were detected in the deutocerebrum and tritocerebrum. Immunoreactive cell processes were observed in the mushroom bodies, the central body, the optic lobes, and in the axon tracts leaving the pars intercerebralis and tritocerebrum. FMRFamide-like material was also seen in the circumoesophageal commissures. Further FMRFamide-like material was present in cell bodies of the suboesophageal ganglion. FMRFamide-like staining activity changed dramatically during the oviposition cycle in mature adult females. The median neurosecretory cells stained lightly immediately after oviposition and remained pale until the third day, when staining of perikarya and axon tracts increased. The staining intensity decreased on days 4 and 5. The titre of FMRFamide-like material in the hemolymph increased during the vitellogenic cycle but plummeted after oviposition. A single band of FMRFamide-like material was evident on immunoblot following sodium dodecyl sulphate-polyacrylamide gel electrophoresis of adult female hemolymph. The approximate molecular weight of this molecule was 8,000. Gel permeation chromatography of hemolymph revealed a FMRFamide-immunoreactive fraction with a molecular weight of 8,000. This fraction possessed myotropic activity when applied to the locust oviduct.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of substitutions at position three on the binding and bioactivity of proctolin in locust hindgut and oviduct.

A number of proctolin analogs modified at position three were analyzed for their relative binding affinities and biological activity on locust hindgut and oviduct. A decrease in chain length at this position (from Leu, Ile to Val) or an increase in hydrophobicity alone (Glu) or combined with a decrease in chain length (Val, Ser, Thr and Asp) decreased bioactivity but not necessarily binding. (Ser3)-proctolin had a higher affinity than proctolin for both hindgut and oviduct membranes but was less biologically active than proctolin in both tissues. Several other analogs bound with a similar affinity to proctolin but were significantly less biologically active, particularly on locust oviduct. These results suggest that the position three leucine of proctolin is more important for bioactivity than for binding in both oviduct and hindgut. The data also suggest the presence of two proctolin receptor subtypes on oviduct but not on hindgut membranes. Position three proctolin analogs may be useful in more precisely distinguishing these subtypes.

The effect of proctolin analogues and other peptides on locust oviduct muscle contractions.

The locust oviduct bioassay system was used to assess the ability of a variety of peptides to induce oviductal contractions. Proctolin analogues were three orders of magnitude less potent than proctolin. Proctolin supra-analogue and Arg-Tyr-Leu-Ala-Thr demonstrated high activity. Perhaps the most significant finding was the discrepancy between the high binding capacity of the proctolin analogue Arg-Tyr-Ser-Pro-Thr and its relatively low myotropic activity. This observation argues for a crucial role for the leucine residue in activating the proctolin receptor. Several other myotropic peptides were tested for their effect on oviduct contractions. FMRFamide caused contractions at doses several orders of magnitude higher than proctolin. The FLRFamide leucomyosuppression inhibited proctolin-induced contractions. In addition, myomodulin and catch relaxing peptide caused oviductal contractions at low concentrations. The enkephalins had no effect when applied alone but potentiated proctolin-induced oviduct contractions. The mechanism of the potentiation is not known. The data argue for the presence of several binding sites on the oviduct membrane.

Pharmacological studies of proctolin receptors on foregut and hindgut of Blaberus craniifer.

Proctolin (Arg-Tyr-Leu-Pro-Thr) and proctolin analogs modified at position 1, 2, or 5 caused dose dependent contractions of Blaberus fore- and hindgut. The varying contractile effects between both tissues revealed the possible presence of receptor subtypes as identified by [GABA1]-proctolin. A single population of binding sites (Kd approximately 100 nM) was deduced from Scatchard analysis. In addition, nanomolar concentrations of proctolin induced a dose-dependent hydrolysis of phosphoinositides (PIns) augmented by GTPgammaS (1 microM) on foregut membranes but no accumulation of cAMP. Proctolin induced contractions are likely mediated via a phospholipase C linked to a heptahelical receptor bound to heterotrimeric G-proteins.

Release of neurosecretory protein from insect neurohaemal tissue following electrical stimulation.

A protein, located within the protocerebral neurosecretory system of locusts, is released from the storage lobes of the corpora cardiaca following the propagation of a compound action potential to the storage lobe via the nervus corpus cardiacum I. Release depends upon the entry of extracellular calcium during depolarization. The data presented here indicate that a protein, similar to the neurophysins of vertebrates, is released from neurohaemal tissue of locusts following stimuli which are known to favour hormone release.

Synaptic transmission elevates adenosine 3', 5'-monophosphate (cyclic AMP) in locust neurosecretory cells.

The release of adipokinetic hormones from the neurosecretory cells within the glandular lobe of the corpus cardiacum (CC) of locusts is under the synaptic control of axons within the nervi corporis cardiaca II (NCC II). This synapse has been shown to be aminergic with the transmitter tentatively identified as octopamine. We show here that stimulation of the presynaptic nerves in NCC II produces an increase in adenosine 3',5'-monophosphate (cyclic AMP) in the glandular lobe. Application of octopamine mimics the effect of nerve stimulation. These and other experiments lead us to believe the increase in cyclic AMP is coupled to the process of synaptic transmission, and that the increase occurs within the intrinsic neurosecretory cells of the glandular lobe.

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.

Utilization of amino acids during development of the African migratory locust embryo.

The metabolic fate of amino acids introduced into the locust egg by the addition of amino acids after oviposition is compared to that of amino acids derived from the maternal haemocoel. It was found that amino acids from the two sources did not always follow the same pattern of utilization. Topical application resulted in some of the applied amino acids being converted into other amino acids and ammonia. When the labelled amino acid was administered to the egg via the maternal haemocoel, radioactivity was restricted to the amino acid orginally provided.

Amino acid composition, degradation and utilization of locust vitellogenin during embryogenesis.

1. The amino acid composition and subunit structure of locust vitellogenin have been investigated. Glycine was the predominant amino acid of the vitellogenin 2. By combining previous data on the free amino acids of the developing egg and on the degradation of the newly characterized vitellogenin, the daily metabolism of amino acids could be calculated. 3. Most amino acids were metabolized extensively at the beginning of embryogenesis. During blastokinesis metabolism slowed then speeded up with the approach of hatching. 4. Serine, glycine and tyrosine demonstrated metabolic activity at variance with this general trend. Serine was the most abundant free amino acid. Presumably much of the glycine released from the vitellogenin contributed to the serine pool. Tyrosine metabolism appeared to be correlated to cuticle sclerotization.

Is octopamine a transmitter mediating hormone release in insects?

The release of hyperlipemic hormone from the glandular cells of the corpus cardiacum (CC) of Locusta migratoria is under the synaptic control of axons in nervus corpus cardiacum II (NCC II). The effects of aminergic agonists and antagonists on the release of the hyperlipemic hormone induced by electrical stimulation of NCC II have been examined. CC isolated from reserpine-injected locusts did not release hormone when subjected to electrical stimulation of NCC II but continued to release hormone in response to high-potassium saline. The electrically stimulated release of hormone from isolated CC was abolished by the alpha-adrenergic blocking agent, phenoxybenzamine, but potentiated by the beta-adrenergic blocking agent, propranolol. Phenoxybenzamine did not interfere with release induced by high-potassium saline. It is suggested that the postsynaptic receptors on the glandular cells are similar to the alpha-adrenergic receptors of vertebrates. Octopamine was found to be present in the glandular lobe of the CC at concentrations of 0.62 pmole per gland pair. Reserpine depleted the content to 0.3 pmole per pair. Bathing the CC in 10(-7) M octopamine resulted in the release of hyperlipemic hormone, and this release was blocked by phenoxybenzamine. It is concluded that the neurotransmitter involved in the synapse between axons of NCC II and the cells releasing hyperlipemic hormone is aminergic, possibly octopaminergic. Octopamine may well be a transmitter mediating hormone release in insects.

A hypoglycemic factor from the corpus cardiacum of the American cockroach Periplaneta americana.

Thin-layer chromatography of cockroach corpora cardiaca (cc) extracts revealed a fraction (F1 in solvent system 1) which had hyperlipemic activity in locusts and which differed in mobility from synthetic locust adipokinetic hormone I (AKH I). Fractions 4 and 5 exhibited hypolipemic activity when injected into locusts. Pooled samples of these fractions caused a diminution of carbohydrate levels in the cockroach. Bovine insulin mimicked both these effects. No change in hemolymph lipid was evident in the cockroach after injection of either CC fractions 4 and 5 or bovine insulin. Both the active fraction from the cockroach CC and bovine insulin caused a decrease in hemolymph carbohydrate in neck-ligated locusts. Injection of locust hypolipemic hormone, separated from locust CC storage lobes by TLC, into neck-ligated locusts also caused a decrease in hemolymph carbohydrate concentration. Normal locusts showed no change in hemolymph carbohydrate.

Insulin-like molecules in the beetle Tenebrio molitor.

Immunocytochemical staining of the nervous system of larva, pupa, and adult stage of Tenebrio molitor with anti-insulin serum demonstrated insulin-like peptides in the protocerebrum, corpora allata, and suboesophageal ganglion. During pupal development, marked changes in staining intensity of the protocerebral cells were detected. The staining pattern suggests release of insulin-like peptides early on day 0 and again on day 3 of the stadium. Injections of anti-insulin at these times caused significant delays in the timing of pupal/adult ecdysis. An immunoblot of haemolymph from day-3 pupae revealed a 6.5-kDa insulin-like molecule. These results suggest that the prothoracicotropic hormone of T. molitor is an insulin-like molecule.

Pharmacology of insecticide-induced release of hyperlipaemic hormone in the locust, Locusta migratoria.

1. The mechanism of insecticide-induced release of hyperlipaemic hormone from the glandular lobe of the isolated corpora cardiaca (CC) of locusts has been studied using pharmacological agents. 2. Treatment of isolated CC with various insecticides induces the release of hyperlipaemic hormone as judged by bioassay. 3. Reserpinisation of CC (25 micrograms/locust) or treatment of isolated CC with the alpha-adrenergic-receptor blocker phentolamine had no effect on the action of DDT or bioresmethrin, but partially blocked the action of dieldrin and chlorfenvinphos. 4. Treatment of isolated CC with the postsynaptic cholinergic blocker hexamethonium bromide abolished the effect of dieldrin and chlorfenvinphos, but did not block the action of DDT or bioresmethrin. 5. The effects of all the insecticides tested in this study were completely blocked by treatment of the CC with 10(-6) M tetrodotoxin. 6. The results indicate that DDT and bioresmethrin may act directly on the glandular cells via a sodium-dependent mechanism. The results with dieldrin and chlorfenvinphos suggest the presence of two distinct cholinergic pathways, one of which acts via the pre-synaptic aminergic terminals which control the glandular cells, whereas the other acts elsewhere in the CC. Sodium channels are also involved in the ultimate expression of these two insecticides.

A FMRFamide-like peptide is associated with the myotropic ovulation hormone in Rhodnius prolixus.

The protocerebral neurosecretory cells previously shown to be the source of the myotropin controlling ovulation in Rhodnius prolixus react in an immunocytochemical assay using an antiserum against FMRFamide. When the same antiserum was injected into fed mated females at the appropriate time the timing of oviposition was delayed, but the total number of eggs developed was unaffected compared to controls injected with pre-immune rabbit serum. The titer of FMRFamide-like peptide (assayed by RIA) in the hemolymph of mated and virgin females was found to fluctuate with the egg laying cycle, and to reflect earlier determinations of the titer of myotropic activity. Western blots of SDS-PAGE revealed a FMRFamide-immunoreactive peptide of approximately 8.5 kDa in both hemolymph and extracts of the ovulation hormone cells.

Insulin-like immunoreactivity and molting in Rhodnius prolixus.

Two prominent cells were observed when fifth stage Rhodnius female larval brains were stained with anti-insulin serum. The staining intensity of these cells varied during the instar, being lowest on Day 1 and Days 5 and 6 after feeding. Injection of anti-insulin serum into 5th stage larvae immediately after feeding and on Days 4 and 5 in females and on Days 5 and 6 in males prevented molting. Control antiserum had no effect on the molting process. Injections at other times during the instar had no effect unless serum was injected just prior to ecdysis. Control or anti-insulin serum injected at this time disrupted normal ecdysis. These results are discussed in terms of the control of the developmental program of the insect.