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.     

Cuticle protein in adult Locusta migratoria

The total protein, chitin and soluble protein content of the abdominal cuticle of male adult locusts was analysed. After an initial period of increase, cuticular protein and chitin content levelled off with the onset of sexual maturation. After ecdysis, the amount of soluble cuticular protein increased unitil maturation. Specific cuticular protein electrophoretic bands decreased in staining intensity with development and were presumed to become bound within the cuticle. Incorporation of ³H-leucine into cuticular proteins was highest into pharate adult cuticle, then decreased after ecdysis to a constant level after maturation. Following sexual maturation when the total cuticular protein and soluble protein content remained constant incroporation of ³H-leucine continued indicating the dynamic nature of the cuticle.     

Ecdysone during ovarian development in Locusta migratoria

Considerable amounts of ecdysteroids are produced during each ovarian cycle in adult females of Locusta when vitellogenesis is almost completed. The hormonal molecules are synthesized at the end of the maturation of the terminal oöcytes during each cycle, at the time when vitellogenesis is almost completed. No synthesis takes place in the absence of ovarian development (allatectomy, ovariectomy), whereas extirpation of the prothoracic glands at the beginning of adult life does not affect ecdysteroid production. More than 95% of the total ecdysteroid content of female adults can be recovered from the ovaries. In vitro studies show that the ovaries produce ecdysteroids and convert labelled cholesterol into ecdysone. Microsurgical experiments indicate that this synthesis takes place in the follicle cells surrounding the oöcyte. The newly synthesized ecdysteroids do not enter massively into the blood, but pass into the oöplasm where they are progressively converted to polar compounds; as a result, at the end of each ovarian cycle, egg-laying corresponds to the disappearance of ecdysteroids from the female insects, the hormonal molecules can easily be recovered from the eggs. A gas chromatographic analysis coupled to mass spectrometry shows that the principal ecdysteroid synthesized by the adult females of Locusta is by far ecdysone. Ecdysterone, the paramount ecdysteroid of the larvae of Locusta, is not present in noticeable amounts in the female adult of this species.     

Excretion of juvenile hormone and its metabolites in the locust, Locusta migratoria

Racemic synthetic ³HC₁₈ juvenile hormone, dissolved in paraffin oil, was injected into adult Locusta migratoria and the excreted radioactive material in the faeces was determined. Within 48 hr two-thirds of the injected radioactivity can be recovered in the frass, half of it within 3 hr. The remaining one-third of the injected label is incorporated or is released as water. Adult locusts of either sex or of different ages show no difference in the metabolic pathways of the JH and its excretion rate.The excreta contain as a degradation product 7-ethyl-3,11-dimethyl-cis-10,11-epoxy-trans, trans-2,6 trideca-dienoic acid, the corresponding dioldienoic acid and the dioldienoic methyl ester. Unchanged Cecropia JH was also found in the frass. The radioactive hormone, as well as the metabolites, were excreted mainly by the Malpighian tubules; smaller amounts of the radioactive material were also found in the fore-, mid, and hindgut.     

The proteases in the gut of the locust, Locusta migratoria

The intestinal fluid of Locusta migratoria was purified by ionexchange chromatography on a DEAE-cellulose column. Four fractions (P_I–P_IV) with endopeptidase activity have been obtained and characterized in further studies. All proteolytic fractions were found to react with PMSF. Therefore, they seem to be typical serine proteases. Two of them, P_I and P_IV, resemble bovine trypsin and bovine chymotrypsin, respectively. These proteases hydrolyse the B-chain of oxidized insulin and the synthetic substrates BTEE,² APNE and BAEE, BANA with a specificity very similar to the bovine enzymes. Moreover, they show similar inhibition characteristics and pH activity profiles. Their molecular weights were found to be 17,000 and 18,200, respectively, according to gel filtration. Fraction P_III did not hydrolyse any of the applied synthetic substrates, P_II was active only with GluPNA. The pH optima of these enzymes lay near neutrality. Their molecular weights were found to be 27,000 and 32,000, respectively. Probably they belong to a type of proteases hitherto scarcely described and not to be found in vertebrates.     

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

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.     

Fate of maternal conjugated ecdysteroids during embryonic development in Locusta migratoria

Newly laid eggs of Locusta migratoria contain impressively high concentrations of conjugated 2-deoxyecdysone and conjugated ecdysone of maternal origin. These molecules are metabolized during embryonic development, the changes concerning not only the ecdysteroid genins but also the conjugating moieties. In the present paper the fates of the maternal conjugates were followed during embryogenesis in the eggs. The conjugates were separated both by silica gel TLC and reverse-phase HPLC and measured, before and after hydrolysis, by RIA. Fluctuations of radioactive ecdysteroid conjugates were also investigated in eggs laid by females subjected to massive injections of tritiated cholesterol. The results are discussed in relation to recent data on identification of ecdysteroid conjugates in Locusta and a model for the sequences of metabolic events leading from maternal ecdysteroid conjugates to the embryonic ecdysteroids is proposed.     

The effects of azadirachtin on the endocrine control of moulting in Locusta migratoria

The effects of azadirachtin, a compound from the neem tree, Azadirachta indica A. Juss, were studied during the last-larval instar of Locusta migratoria. A dose-response relationship was established using moult inhibition and mortality as effective parameters. Although injected azadirachtin elicits feeding inhibition, our results prove that moult inhibition is due to an interference with the endocrine system rather than to the altered feeding behaviour. Modification and suppression of the ecdysteroid titre by azadirachtin is closely correlated with the morphogenetic effects. Inhibition of eclosion processes, however, suggest a wide-spread blockage of factors presumably located in the central nervous system.     

Precocene II has juvenile-hormone effects in 5th instar Locusta migratoria

Precocene II can cause juvenilization of 5th instar Locusta migratoria. The adultoids and supernumerary larvae produced following precocene treatment are morphologically indistinguishable from adultoids produced by treatment with a JH-analogue, while the CA are markedly reduced in size or absent. The time-response curves for the induction of juvenile characters and for the degeneration of the CA suggest that the CA may be active during the early 5th instar. The CA are necessary for juvenilization to occur in response to precocene, and the effect is probably the result of synthesis or release of JH during the breakdown of the glands.     

Neural substrate and allatostatin-like innervation of the gut of Locusta migratoria

Allatostatin-like immunoreactivity (ALI) is widely distributed in processes and varicosities on the fore-, mid-, and hindgut of the locust, and within midgut open-type endocrine-like cells. ALI is also observed in cells and processes in all ganglia of the central nervous system (CNS) and the stomatogastric nervous system (SNS). Ventral unpaired median neurons (VUMs) contained ALI within abdominal ganglia IV-VII. Neurobiotin retrograde fills of the branches of the 11th sternal nerve that innervate the hindgut revealed 2-4 VUMs in abdominal ganglia IV-VIIth, which also contain ALI. The VIIIth abdominal ganglion contained three ventral medial groups of neurons that filled with neurobiotin and contained ALI. The co-localization of ALI in the identified neurons suggests that these cells are the source of ALI on the hindgut. A retrograde fill of the nerves of the ingluvial ganglia that innervate the foregut revealed numerous neurons within the frontal ganglion and an extensive neuropile in the hypocerebral ganglion, but there seems to be no apparent co-localization of neurobiotin and ALI in these neurons, indicating the source of ALI on the foregut comes via the brain, through the SNS.     

Infection of Locusta migratoria with entomopoxviruses from Arphia conspersa and Melanoplus sanguinipes grasshoppers

Entomopoxvirus (EPV) occlusion bodies isolated from Arphia conspersa and Melanoplus sanguinipes grasshoppers were fed to 3rd and 4th instar Locusta migratoria nymphs. Locus mortality induced by A. conspersa EPV was first detected 18 days after addition of virus to the diet, and reached a level of approximately 68% of the colony population by 60 days after virus inoculation. In a similar population of L. migratoria nymphs, mortality induced by M. sanguinipes virus reached 90% 60 days after virus inoculation. Entomopoxvirus was isolated from M. sanguinipes EPV infected locust nymphs and the viral DNA was cleaved with several restriction endonucleases. The DNA fragment patterns obtained after agarose gel electrophoresis were compared with the fragment patterns from the original sample of M. sanguinipes EPV DNA cleaved with the same restriction endonucleases. No differences in the cleavage patterns were detected between the two virus DNA samples. Virus structural proteins of M. sanguinipes EPV purified from infected locust nymphs were compared by polyacrylamide gel electrophoresis with virus proteins isolated from the original sample of M. sanguinipes EPV. A total of six different virus protein bands were detected between the two poxvirus preparations.     

Ecdysone titre and metabolism in relation to cuticulogenesis in embryos of Locusta migratoria

Eggs of Locusta migratoria contain remarkably high concentrations of ecdysone and several other ecdysteroids. During the time-span of embryonic development (11 days) 4 distinct peaks of ecdysone concentration (up to 8 μM) are observed in the egg, demonstrating the ecdysiosynthetic capacity of the embryo. Only during postblastokinetic development, is ecdysone efficiently hydroxylated to 20-hydroxyachieved through conjugation. On the basis of optical and electron microscopic observations, we have been able to correlate precisely each of the four peaks of ecdysone concentration in the egg with the time of deposition of a cuticle by the embryonic tissues (peak 1: serosal cuticle; peak 2: first embryonic cuticle; peak 3: second embryonic cuticle; peak 4: third embryonic cuticle).     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body: Cytological development

Cytological development in the fat body of adult female Locusta migratoria, related to vitellogenin synthesis, has been studied by light and electron microscopy. In the newly-emerged adult, the cells are filled with lipid droplets, which indent the nucleus, and with fields of glycogen, while ribosomes and endoplasmic reticulum are scarce. Correlated with the onset of vitellogenin synthesis, about day 8 of adult life, the nucleus enlarges, lipid droplets and glycogen decrease, and rough endoplasmic reticulum and Golgi complexes become the most abundant organelles. These changes reflect a conversion of the principal role of the fat body from nutrient storage to the synthesis and secretion of protein. They are prevented by allatectomy, and restored by subsequent treatment with the juvenile hormone analogue, ZR-515. Late in the first gonotrophic cycle, about day 20, dense bodies, vesicle-containing bodies and lysosomes are seen, indicating recycling of cellular materials. Five days after ZR-515 treatment, when protein synthesis has declined, the rough endoplasmic reticulum appears in arrays adjacent to lipid droplets, possibly awaiting reactivation. By the use of ferritin-labelled antivitellin immunoglobulin, vitellogenin has been localized intracellularly in the RER saccules and Golgi vesicles, and extracellularly in channels between the folded plasma membranes, showing sites of accumulation and secretion of this protein.     

A carrier lipoprotein for juvenile hormone in the haemolymph of Locusta migratoria

In the haemolymph of adult female locusts six different lipoprotein fractions have been demonstrated by means of isoelectric focusing. One of these binds injected ³H-Cecropia juvenile hormone. The carrier protein is a yellow lipoprotein with a molecular weight of approximately 220,000 daltons and an isoelectric point of pH 6·8. The binding of the hormone to the protein is stable during gel filtration over Sephadex G-25 and during dialysis for 24 hr against phosphate buffer pH 7·0.The hormone is quickly metabolized in the locusts. In the haemolymph were found more polar compounds such as 10-epoxy-7-ethyl-3,11 dimethyl-2,6-tridecadienoic acid and the corresponding dioldienoic acid.Both compounds were not bound by the pH 6·8 carrier lipoprotein under in vivo conditions.     

Dynamics of energy substrates in the haemolymph of Locusta migratoria during flight

In the two-fuel system for flight of the migratory locust, the haemolymph carbohydrate concentration falls during flight periods of up to 1 hr, the decrease being greater in case the pre-flight carbohydrate level is higher. The increase in the lipid concentration from the onset of flight is virtually independent of the initial lipid concentration. Flight intensity affects these changes in substrate concentrations: the carbohydrate level decreases more rapidly if flight speed is higher, whereas the increase in lipid concentration is delayed at higher flight speeds. Respiratory carbon dioxide production is elevated rapidly during flight and reaches over eight times the resting level. From the rate of ¹⁴CO₂ production after labelling of the haemolymph diglyceride pool it is concluded that diglycerides contribute to providing the energy for flight from the earliest stage of flying activity; diglyceride oxidation increases until maximum utilization is attained after some 45 min of flight. The decline in haemolymph carbohydrate concentration due to flying activity results in a decrease of haemolymph osmolarity. Free amino acids, particularly taurine, increase markedly in the haemolymph during flight; yet their concentration only partially counterbalances the fall in haemolymph osmolarity.     

Neurosecretory protein synthesis in relation to starvation and feeding in adult male Locusta migratoria

An immunological approach has been used to assess the effects of starvation and subsequent feeding upon the synthesis of neurosecretory protein in adult male Locusta migratoria. Starvation (for 5 days) did not reduce the incorporation of radioactive cystine into neurosecretory protein. However, the rate of neurosecretory transport in starved insects was approximately half that in normal fed insects.Within 1 hr after the start of feeding the rate of neurosecretory protein synthesis more than doubled and the rate at which newly synthesized protein was transported to the CC increased approximately three-fold. The incorporation of cystine into specific (i.e. neurosecretory) protein was always higher, even in starved insects, than into the non-specific proteins (i.e. proteins other than those from the A-cells) of the brain. This difference was maximal at 4 hr after feeding when the incorporation into specific protein was more than 5 times that into non-specific protein.     

Feeding impairs chill coma recovery in the migratory locust (Locusta migratoria)

Low temperature causes loss of neuromuscular function in a wide range of insects, such that the animals enter a state known as chill coma. The ability to recover from chill coma (chill coma recovery time) is often a popular phenotype to characterise chill tolerance in insects. Chill coma in insects has been shown to be associated with a decrease in haemolymph volume and a marked increase in [K⁺], causing dissipation of K⁺ equilibrium potential and resting membrane potential. High potassium diet (wheat) has also previously been shown to increase haemolymph [K⁺] in Locusta migratoria leading to sluggish behaviour. The present study combined these two independent stressors of ion and water homeostasis, in order to investigate the role of K⁺- and water-balance during recovery from chill coma, in the chill sensitive insect L. migratoria. We confirmed that cold shock elicits a fast increase in haemolymph [K⁺] which is likely caused by a water shift from the haemolymph to the muscles and other tissues. Recovery of haemolymph [K⁺] is however not only reliant on recovery of haemolymph volume, as the recovery of water and K⁺ is decoupled. Chill coma recovery time, after 2 h at −4 °C, differed significantly between fasted animals and those fed on high K⁺ diet. This difference was not associated with an increased disturbance of haemolymph [K⁺] in the fed animals, instead it was associated with a slowed recovery of muscle [K⁺], muslce water, haemolymph [Na⁺] and K⁺equilibrium potential in the fed animals.     

Stimulated fluid secretion is sodium dependent in the Malpighian tubules of Locusta migratoria

The ionic dependencies of stimulated and unstimulated Locusta tubules have been studied. K⁺, Na⁺, Cl^? are essential to both basal and stimulated secretion. K⁺ is secreted against a concentration gradient in unstimulated tubules. In response to diuretic hormone or cAMP application, there is a dramatic influx of K⁺ into the lumen. A high level of Na⁺ and Cl^? in the bathing medium is required to allow maximal fluid secretion. The tubules show an apparent impermeability to Na⁺; its concentration in the secreted fluid is always much less than in the bathing medium. If Na⁺ is omitted from the medium and excess K⁺ added (80 mM K), then although basal secretion occurs (2.5 nl/min), the tubules fail to respond to stimulation. Clearly Na⁺ has an important indirect role to play in stimulated fluid secretion.     

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.     

Phagocytosis of inert particles in Locusta migratoria and Galleria mellonella: Study of ultrastructure and clearance

Inert particles (iron saccharate or latex beads) injected in the haemocoel of Locusta migratoria, are taken up by pericardial cells (iron saccharate only), reticular cells of the haemopoietic tissue and certain haemocytes: plasmatocytes and coagulocytes; these two haemocyte types are also the main phagocytic blood cells in Galleria mellonella.Necrosis of phagocytic haemocytes, following injection of an overdose of iron saccharate, explains the profound modifications of the haemogram observed during the first 24 hr following injection; the macrophagic evolution of reticular cells slows down the haemopoietic differentiation of these cells and explains the long term disturbances of the blood picture.Clearance of latex beads injected in larvae of Locusta complies to an exponential function of time; we can determine a granulopectic index which will permit comparisons to be made between clearance of inert and of ‘antigenic-like’ particles.