Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria)

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.     

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.     

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.     

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.     

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.     

Soft-tissue accumulation of lead in the blue tilapia,Oreochromis aureus (steindachner), and the modifying effects of cadmium and mercury

The interaction of mercury and cadmium with lead was investigated by exposingOreochromis aureus to two heavy metals simulataneously. The chronic accumulation prolife of lead was determined by analyzing the liver, brain, gill filaments, intestine, caudal muscle, spleen, trunk kidney, and gonads following exposure to lead alone and in mixtures with mercury and cadmium. Nominal exposure concentrations of lead were 0.05, 0.10, 0.50, and 1.00 mg/L. Mixtures of lead (0.50 or 0.05 mg/L) with cadmium (0.05 mg/L) and lead (0.50 or 0.05 mg/L) with mercury (0.05 mg/L) were also used. Following 140 d of exposure to lead, the highest concentrations of lead consistently accumulated in the trunk kidney. The concentration of lead in the kidney was decreased by coexposure to mercury or cadmium, but increased in the muscle and liver. Under all exposure regimes, the median concentration of lead in the muscle exceeded safety levels recommended for human consumption. In a food fish, such asO. aureus, a knowledge of toxic metal accumulation patterns is of great importance.     

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.     

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.     

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.     

Activity of disconnected corpora allata in Locusta migratoria: Juvenile hormone biosynthesis in vitro and physiological effects in vivo

Severance of nervi corporis allati I (NCA I) in day-1 adult female Locusta migratoria resulted in a significant decrease and a loss of the characteristic pattern of juvenile hormone biosynthesis by the corpora allata as determined by radiochemical assay. This decrease in the rate of juvenile hormone biosynthesis was not reflected in basal oöcyte growth. The lengths of the oöcytes were the same in NCA-transectioned and in the sham-operated females. The effect of severance of both NCA I and NCA II on juvenile hormone biosynthesis and ovarian maturation was similar to the effect of NCA I severance only.Rate of juvenile hormone biosynthesis by corpora allata of fourth-instar larvae exhibited a maximum of activity in the middle of the stadium. The severance of NCA I early in the stadium resulted in a very low rate of juvenile hormone biosynthesis and a disappearance of this peak. In NCA I-transectioned larvae, the duration of the stadium was significantly increased although larvae moulted into normal fifth instar.     

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).     

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.     

Vitellogenin in locusts (Locusta migratoria): Translation of vitellogenin mRNA in Xenopus oocytes and analysis of the polypeptide products

Cytoplasmic poly(A)⁺-RNA, prepared from fat bodies of reproductively active locust females, directed the synthesis of two large polypeptides in Xenopus oocytes. Occasionally two smaller polypeptides (X₁ and X₂) were also detectable. The two larger polypeptides were immunologically and electrophoretically indistinguishable from the unprocessed Vitellogenin polypeptides (Vg₁ and Vg₂) of locust fat bodies. Peptide patterns generated from these two translation products by Staphylococcus aureus V8 protease digestion were identical to those of Vg₁ and Vg₂. RNA isolated from allatectomized female locusts treated with the juvenile hormone analog (ZR-515) was also able to direct the synthesis of vitellogenin in Xenopus oocytes, whereas RNA isolated from mature males or allatectomized females did not. The molecular weights of fat-body Vg₁ and Vg₂ were 235,000 and 225,000, respectively and the processed vitellogenin polypeptides were found to range from 126,000 to 52,000. Electrophoretic and Chromatographic analysis of [³⁵S]methionine-containing tryptic peptides of Vg₁ and Vg₂ showed two different tryptic peptide fingerprints. Distinctly different peptide patterns were also observed when Vg₁ and Vg₂ were partially digested with V8 protease or papain. However, tryptic peptide mapping and V8 protease limited digestion mapping of fat-body X₁ and X₂ revealed that these two polypeptides were derived from Vg₁ and Vg₂. This suggests that Vg₁ and Vg₂ are products of two different vitellogenin structural genes.     

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.     

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.     

Identification of pheromone-like compounds in male reproductive organs of the oriental locust Locusta migratoria

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.     

Vitellogenin accumulation in the fat body and haemolymph of Locusta migratoria in relation to egg maturation

Vitellogenins first appear in the fat body of Locusta migratoria during subphase I of vitellogenesis and increase to a constant level during subphase II. A second increase occurs shortly before the oöcyte attains maximal size. Vitellogenin content of fat body subsequently returns to that of subphase I, appropriate to the size of the subterminal oöcyte. The absolute amount of vitellogenin in the fat body is low compared to that found in the haemolymph. Fat body and haemolymph vitellogenins have immunological properties similar to oöcyte yolk proteins—when challenged with oöcyte protein antiserum. They exhibit similar electrophoretic mobility in polyacrylamide gel electrophoresis and are complex glyco-lipoproteins.     

Moulting and ecdysone release in response to electrical stimulation of protocerebral neurosecretory cells in Locusta migratoria

Locusta migratoria larvae were submitted to electrical stimulation of the protocerebral neurosecretory cells (median neurosecretory cells of the pars intercerebralis and lateral neurosecretory cells), during the last larval instar. The effects of the treatment were observed both on the duration of the stage and on the variations in haemolymph ecdysone levels. In untreated larvae, there was an initial ecdysone peak at the beginning of day 5, which was followed by 4 larger peaks between days 6 and 8. Stimulation of the median neurosecretory cells at the beginning of the instar resulted in the formation of one very large hormonal peak at the end of day 3: a day and a half earlier than in the control groups. Moulting was likewise accelerated. Stimulation also increased the size of the peaks, as compared with the controls. Stimulation of the lateral neurosecretory cells had a weaker ecdysiotropic effect; neither the number nor the size of the peaks were changed, though, like ecdysis, they occurred earlier. Stimulation of the deutocerebrum had no effect on either ecdysone titres or moulting. Electrical stimulation of the median neurosecretory cells at the end of day 5, that is after the occurrence of the first ecdysone peak, shortened the larval stage while having no significant effect on ecdysone levels in the haemolymph. The neuroendocrine control of ecdysis in Locusta is discussed.