Effect of high potassium concentrations on juvenile hormone release in vitro from corpora allata of Locusta migratoria

ABSTRACT. Incubation of corpora allata (CA) from adult females of Locusta migratoria (L.) in vitro in medium with a potassium concentration of 50 mM results in an elevation of the rate of juvenile hormone (JH) release. This elevation is however delayed, becoming apparent after the glands have been returned to low concentration of potassium but it is also prolonged, lasting up to 270 min. The elevation is initially modest but becomes more marked with time. The response of glands to high concentration of potassium is heterogeneous and appears to some extent to be a function of the initial rate of JH release of the glands. Glands starting with low rates of JH release are stimulated strongly by potassium, those with high rates of JH release much less so. Co-incubation of glands with high concentration of potassium and with calcium channel blockers (both organic and ionic) prevents the elevation of release rates otherwise consequent on high concentrations of potassium.     

An allatostatic factor and juvenile hormone synthesis by corpora allata in Locusta migratoria

The hemolymph juvenile hormone (JH) titer in sexually immature female adults of Locusta migratoria (Ibaraki strain, Japan) was lower than in sexually mature females; nevertheless, JH synthetic activity by the corpora allata (CA) in vitro was considerably higher in immature females than in sexually mature females ([Okuda et al., 1996]). We carried out experiments to explain this contradiction. The CA activity of sexually immature female adults was very low when the CA were incubated as a complex together with the corpora cardiaca (CC) and brain. When the same complex was assayed after cutting the nerve cord connecting the CC and CA (NCA1), JH synthesis by the CA was enhanced tenfold. When this pair of CA was incubated in fresh medium without the CC and brain, JH synthesis was further increased. Therefore, the higher in-vitro JH production by CA from immature female adults was the result of isolation of the CA from the brain and CC. A methanolic extract of brain-CC complexes contained a factor that inhibited JH synthetic activity by CA in vitro in both immature and mature insects, and this inhibition was reversible. The factor was heat-resistant but lost allatostatic activity after pronase digestion. These results indicate that the allatostatic factor is probably a heat-stable peptide.     

Juvenile hormone esterase activity from Manduca sexta corpora allata in vitro

Juvenile hormone esterase (JHE) activity released by the corpora allata (CA) into incubation media (CA-JHE) was titered daily during the course of the last (fifth [V]) larval stadium of Manduca sexta. This CA-JHE activity was relatively low during the early last stadium up to the time of commitment (V4), then rose rapidly to a peak on V6. Activity declined sharply almost to precommitment levels by V8, before rising to a second peak on the first day of the pupal phase (P0). This pattern of activity is distinct from that of hemolymph JHE activity, which peaks just prior to wandering on V4 and again just prior to pupation (V9). Although the CA-JHE and hemolymph-JHE possess different temporal patterns of activity, isoelectric focusing, gel electrophoresis, and initial studies with selected inhibitors suggest that the enzymes responsible for the CA-JHE and hemolymph-JHE activities are similar, but not identical, in nature. Exposure of the V6 CA in vitro to JH II (0.1 μM) or fluoromevalonolactone (FMev; 0.1 mM) produced an approximate fivefold increase and 60% decrease in JH acid synthesis, respectively. Conversely, the same treatments resulted in an inhibition (JH II) and stimulation (FMev) of CA-JHE activity. These observations suggest that JH may be involved in the direct positive feedback regulation of postwandering larval CA and that the CA-JHE may also be integrally related to this positive feedback mechanism.     

Precocene-induced necrosis and haemocyte-mediated breakdown of corpora allata in nymphs of the locust Locusta migratoria

Light and electron microscopic studies, including the use of the vital dyes trypan blue and acridine orange, indicate that the topical application of precocene II rapidly triggers degenerative processes in the corpora allata (CA) leading within a few days to the virtual disappearance of the parenchymal cells of these glands. The following sequence of events was observed: 1) Within 2 h, many of the cells in fixed nymphal specimens showed increased electron density. During the next few hours, they decreased considerably in volume (shrinkage necrosis). Intercellular spaces increased simultaneously. Although a variable number of cells remained electron lucent, they showed nuclear and cytoplasmic degeneration later on (coagulative necrosis). 2) Haemocytes arrived at the CA sheath and invaded the CA in large numbers after 12 h. 3) CA cells became increasingly necrotic, and cell fragments were budded off and were phagocytosed most noticeably after 1 to 3 days. Thereafter no CA parenchyma remained. 4) Haemocytes dispersed.     

The activity of denervated corpora allata in a diapausing strain of Locusta migratoria: in vivo and in vitro studies

Summary

The activity of denervated corpora allata of a diapausing strain of Locusta migratoria is compared in vivo and in vitro. Inactive corpora allata (CA) of diapausing adult females soon become active following implantation into other diapausing females. Activation also follows incubation in vitro, although this activity decreases rapidly. Active CA of animals whose diapause has been broken by NCC2 section continue to secrete hormone when implanted into host animals (i.e., in vivo) although activity declines after 3 days. In contrast such CA show very low activity in vitro. These data confirm that in L. migratoria activity of the CA is regulated by a dual control involving inhibiting and activating factors whose origins are discussed.     

Morph-specific diurnal variation in allatostatin immunostaining in the corpora allata of Gryllus firmus: Implications for the regulation of a morph-specific circadian rhythm for JH biosynthetic rate

Previous studies have documented a circadian cycle in juvenile hormone (JH) biosynthesis in the long-winged, flight-capable morph, but not in the short-winged flightless morph of the cricket Gryllus firmus. One rapid and reversible inhibitor of in vitro JH biosynthesis by the corpora allata (CA) in crickets is the neuropeptide Phe-Gly-Leu/Ile-amide type of allatostatins (ASTs). To investigate the possible role of allatostatin regulation of the morph-specific circadian cycle of JH production, the quantity of this type of AST in the nerves within the CA was determined by the density of anti-AST-immunostaining in confocal images using the Image J program. The density of immunostaining was inversely related to the rate of JH biosynthesis: Immunostaining in the CA was high and did not differ between morphs early in the photophase when the in vitro rate of JH biosynthesis is low and equivalent in the morphs. However, during the end of the photophase, when the rate of JH biosynthesis rises dramatically in the flight-capable morph, but not in the flightless morph, immunostaining was significantly lower in the flight-capable compared to the flightless morph. These results indicate that morph-specific differences in delivery of AST to the CA and its probable release likely regulate the morph-specific circadian pattern of JH biosynthesis. Also, the negative correlation between AST density and JH production provides evidence for predicting the periods of altered release of these rapid-acting paracine regulators of JH biosynthesis.     

Flight orientation of swarming Locusta migratoria

ABSTRACT. High-speed films of four swarms of Locusta migratoria in Australia and one swarm in New Guinea were analysed. Measurements were made of the locust's body orientation and flight track in the horizontal relative to wind direction, and of height and speed of flight. In all swarms mean course angle and mean track angle in relation to wind direction were significantly different from zero, although all indicated an upwind direction. No evidence was found for orientation to compass direction or sun. Considerable fluctuations in flight direction were measured in some individuals as they traversed the field of view. A modification of Kennedy's (1951) theory is adopted to explain the angled orientations to wind. It is suggested that this could be the result of an optical orientation mechanism.     

Characterization of juvenile hormone-binding proteins of hemolymph of Locusta migratoria

The binding of juvenile hormone (JH) by components from hemolymph of adult female Locusta migratoria was characterized to establish whether hemolymph JH-binding proteins could be distinguished from a protein of fat body (BP-1) that may be a JH receptor. Hemolymph was analyzed by the hydroxyapatite assay, gel separation chromatography, polyacrylamide gel electrophoresis, and density gradient centrifugation. Three fractions that bound JH were separated from whole hemolymph by DEAE cellulose column chromatography, and these differed from all three cytosol-binding components. The major hemolymph component (H-A) showed relatively stable binding of JH, a slight loss of binding capacity after delipidation, and a K_d for JH-I of 16 nM. The K_ds for JH-l and JH-lll with unfractionated hemolymph were 26 and 42 nM respectively. The order of effectiveness of competitors for binding of [³H]JH-l was JH-lll > JH-l ? methoprene > hydroprene ? acids of methoprene and hydroprene. The data indicated that unlabeled JH-lll was bound more effectively than its radioactive counterpart. The sedimentation values determined by sucrose density gradient ultracentrifugation were 13-14 S for hemolymph, and the sedimentation value was not altered by the inclusion of 0.4 M KCl throughout the gradient. The data indicated that H-A resembled the specific JH carriers and differed from the putative receptor of fat body cytosol by several criteria.     

Chemo-discriminatory neurones in the sub-oesophageal ganglion of Locusta migratoria

The sub-oesophageal ganglion of Locusta migratoria was searched for neurones responsive to stimulation of the maxillary palps using intracellular recording techniques. Two plant stimuli were used: wheat, a host plant and cabbage, an unacceptable non-host plant. The stimuli were presented to the palp as both intact leaf tissue and as droplets of aqueous solutions of plant extracts. All the sampled neurones that responded to stimulation of the palp also responded to simple mechanical stimulation. However, 25% of the neurones exhibited consistent differences in response to the two plants when presented as both leaf tissue and droplets, strongly suggesting that they also received a chemosensory input. These differential responses most commonly took the form of differences in the duration of cell activity and/or variation in the latency of the onset of response. The receptive fields of differentially responding neurones were confined to the maxillary palp, or at most to the maxillary and labial palps.     

Noise filtering in the auditory system of Locusta migratoria L

Many acoustically communicating grasshoppers live in crowded populations where sound of many individuals may cause permanent noise. Tympanic receptors and first-order auditory interneurons of Locusta migratoria code such noise tonically, whereas many higher order interneurons react only weakly. In response to simultaneously presented sound they exhibit a better signal-to-noise ratio than their presynaptic elements. Two possible filter mechanisms are suggested for noise reduction in higher-order interneurons: (i) high-pass filtering of receptor spike frequencies and (ii) filtering due to synchronization of receptor spikes. Different receptor spike frequencies were elicited by series of short noise pulses with variable repetition rates. Receptor activities differing in their degree of synchronization were elicited by sound stimuli with variable rising times. In contrast to the first order interneurons some higher order interneurons responded best to receptor spike frequencies above 150–200 Hz, thus showing the postulated filtering. Only one higher order interneuron (AN4) distinguished between synchronous and asynchronous receptor activities. It is suggested that high-pass filtering of receptor spike frequencies is responsible for the noise filtering observed in these interneurons. The synchronization selectivity of AN4 is proposed to be responsible for temporal pattern detection of conspecific sounds.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

The absolute configuration of precocene I dihydrodiols produced by metabolism of precocene I by corpora allata of Locusta migratoria, in vitro

The corpora allata from adult female Locusta migratoria metabolize precocene I (7-methoxy-2,2-dimethyl-2H-benzo [b]pyran to cis- & trans-precocene I dihydrodiols (3,4-dihydro-7-methoxy-2,2-dimethyl-2H-benzo [b]pyran-3,4-diol). Derivatization of the dihydrodiols with (-)menthoxy acetyl chloride allowed complete resolution of all four optical isomers. When [4-3H]-precocene I was incubated in vitro with Locusta migratoria corpora allata, it was metabolized stereospecifically to (-)trans-(3R,4S) and (+)cis-(3R,4R) dihydrodiols. Approximately half the precosyl residues bound to cellular macromolecules were discharged by heating to 95 degrees C at neutral pH, as dihydrodiols of the same stereochemistry.     

Stress-induced thermotolerance of ventilatory motor pattern generation in the locust, Locusta migratoria

Ventilation is a crucial motor activity that provides organisms with an adequate circulation of respiratory gases. For animals that exist in harsh environments, an important goal is to protect ventilation under extreme conditions. Heat shock, anoxia, and cold shock are environmental stresses that have previously been shown to trigger protective responses. We used the locust to examine stress-induced thermotolerance by monitoring the ability of the central nervous system to generate ventilatory motor patterns during a subsequent heat exposure. Preparations from pre-stressed animals had an increased incidence of motor pattern recovery following heat-induced failure, however, prior stress did not alter the characteristics of the ventilatory motor pattern. During constant heat exposure at sub-lethal temperatures, we observed a protective effect of heat shock pre-treatment. Serotonin application had similar effects on motor patterns when compared to prior heat shock. These studies are consistent with previous studies that indicate prior exposure to extreme temperatures and hypoxia can protect neural operation against high temperature stress. They further suggest that the protective mechanism is a time-dependent process best revealed during prolonged exposure to extreme temperatures and is mediated by a neuromodulator such as serotonin.     

Ultrastructure of the muscles of the dorsal diaphragm in Locusta migratoria

Summary The alary muscles of Locusta migratoria adults make up the major tissue of the dorsal diaphragm which separates pericardial and perivisceral sinuses in the abdomen. The alary muscles are striated with a sarcomere at rest measuring about 9 m. The Z-line has a staggered-beaded arrangement with A-bands and I-bands readily discernable. Thick myofilaments are surrounded by 10 or more thin filaments. The sarcoplasm has few mitochondria near the area of the Z-line, dyads are present and sarcoplasmic reticulum is poorly developed. Axons which innervate the alary muscle are either contained within invaginated folds of the sarcolemma of the muscle cells or the muscle cells send finger-like projections to envelop the axons. The synaptic terminals contain synaptic vesicles between 40 and 45 nm in diameter and a few electron-dense granules near or less than 170 nm in diameter. Away from synaptic terminals the axon profiles show few or no granules. The axons are accompanied everywhere by well-developed glial cells. This then is not typical neurosecretomotor innervation, however, the presence of electron-dense granules suggests the possibility of peptidergic neurotransmission.     

Ultrastructural localization of juvenile hormone biosynthesis by insect corpora allata

Summary The conversion of exogenous ³H-farnesenic acid to ³H-methyl farnesoate and ³H-C₁₆ juvenile hormone (JH) has been followed in the corpus allatum (CA) cells of the desert locust Schistocerca gregaria by means of electron microscopic autoradiography. Aerobic and anaerobic chase incubations have been used to modify the quantities of these three compounds within the CA cells. Under all incubation conditions, radiolabel is found associated almost exclusively with the subcellular membrane systems — smooth endoplasmic reticulum (SER) and Golgi elements —and with the mitochondria. CA cells are probably similar to vertebrate steroid-synthesizing cells in that the secretory product is synthesized in the SER and mitochondria.Radiolabel was found to be present in all cells of the CA suggesting that all cells are capable of at least the final two stages of JH biosynthesis (the esterification and epoxidation of ³H-farnesenic aid). This indicates that JH biosynthesis may be regulated through changes in the biosynthetic capabilities of individual cells rather than through changes in the total number of cells engaged in biosynthesis. Radiolabel was not observed to be associated with any distinctive cellular product, a result which provides additional evidence for the suggestion that the release of JH from the CA is governed by laws of simple physical diffusion.Supported by operating grants from the National Research Council of Canada to SST and ASMS. ³H-farnesenic acid was supplied by the late Dr. A.F. White of the Unit of Invertebrate Chemistry and Physiology, A.R.C., University of Sussex. We thank Dr. G.E. Pratt for helpful discussions     

The structure of bimodal chemo-, thermo-, and hygroreceptive sensilla on the antenna of Locusta migratoria

Summary Coeloconic sensilla on the antenna of Locusta migratoria were investigated electrophysiologically, labeled and then examined under the scanning or transmission electron microscope. They can be categorized into two main morphological types: 1) sensilla with wall pores and two concentric cuticular walls (wp-dw sensilla), and 2) sensilla lacking wall pores (np-sensilla).Hygroreceptors were observed only in np-sensilla. Olfactory receptors were observed in wp-dw sensilla, but not in np-sensilla. Cold-sensitive units were found in both types. Wp-dw sensilla with a cold-sensitive unit also manifest a fatty acid olfactory receptor. This cold-sensitive unit appears to be less sensitive to drops in temperature than the cold receptor combined with hygroreceptors in np-sensilla. Recordings from both types revealed up to three or sometimes four units that could be distinguished on the basis of impulse amplitude.The number of np-sensilla on each antennal segment ranges from 0 to 6. These sensilla are pegs 4–5 m long with a terminal swelling. The dendritic outer segments of two sensory cells fit tightly into these pegs. The dendrite of the third sensory cell ends at its base. All three are up to 1 m thick, unbranched, and densely filled with microtubules. The cuticular wall and socket of the peg are characterized by (1) an electron-dense inner layer surrounding the dendrites and continuous with electron dense clusters at the base of the peg, and (2) clefts in the cuticle at the insertion of the peg.The present observations favor the hypothesis that insect sensilla combining the triad of moist air, dry air and cold receptors generally lack pores. Specifical, though still alternative roles in stimulus transduction can be suggested for substructures demonstrated in the present and previous electron microscopic investigations.Supported by the Deutsche Forschungsgemeinschaft (SFB 4:G1/D)     

亚洲飞蝗发声器结构与鸣声时域特征研究

本文对亚洲飞蝗Locusta migratoria migratoria(L.)发声器的结构及鸣声的时域特征进行了研究.亚洲飞蝗主要的发声方式为前翅中闰脉-后足股节型,即前翅中闰脉的发声齿与后足股节内侧隆线相互摩擦发出鸣声.应用扫描电子显微镜技术对发声器的结构进行观察,结果表明,亚洲飞蝗前翅中闰脉具有单排规则排列的发声...     

The vasopressin-like immunoreactive (VPLI) neurons of the locust,Locusta migratoria. II. Physiology

Summary The two vasopressin-like immunoreactive (VPLI) neurons of the locust, Locusta migratoria, have cell bodies in the suboesophageal ganglion and extensive arborizations throughout the CNS. One of the two peptides responsible for AVP-like immunoreactivity is a vasopressin-related peptide with putative diuretic hormone properties. These neurons also have FLRF-like immunoreactivity, probably due to the FMRF-amide-related peptide, SchistoFLRF-amide, isolated from Schistocerca gregaria. This peptide has cardioinhibitory activity and a dual potentiation/inhibition of slow motoneuron induced muscle-twitch tension. Although haemolymph AVP-like peptide titre fluctuates under various conditions, the mechanism that regulates neurohaemal release of this peptide is not understood. Very little is known of the release of SchistoFLRF-amide. We have used intracellular recording from VPLI neurons in vivo to reveal synaptic inputs that lead to changes in their level of spiking activity, and probably, release of both the AVP-like peptides and SchistoFLRF-amide. This pair of neurosecretory cells has a major, common excitatory input whose sustained rate of activity is inversely related to light intensity; VPLI spiking activity, driven by this input, is greater in the dark than in light. This input is from a pair of descending brain interneurons. Their light-sensitivity persists after ablation of compound eyes, optic lobes and ocelli, showing them to be part of an extra-ocular photoreceptor system. Attempts to record from, and individually stain, the descending neuron have been unsuccessful, although its axon location and diameter in the circumoesophageal connective have been determined. Possible locations for its cell body have been identified; one region, close to the pars intercerebralis, is known to be photosensitive in some insects. Mechanosensory stimuli also lead to brief increases in VPLI spiking activity via the descending interneuron, though this modality rapidly habituates. We detect no changes in VPLI spiking activity that consistently correlate with the osmolality of perfusion salines; such changes might have been expected from their previously proposed role in water homeostasis. Alternative roles for VPLI cells are discussed.Abbreviations AVP arginine-vasopressin - EOP extra-ocular photoreceptor - FLRF Phe-Leu-Arg-Phe - FMRF-amide Phe-Met-Arg-Phe-amide - RH relative humidity - RIA radio-immune assay - SOG suboesophageal ganglion - VPLI vasopressin-like immunoreactive     

Pléomorphisme mitochondrial dans les corpora allata de Locusta migratoria migratorioides (R. et F.) au cours de la vie imaginale

Résumé Dans les corpora allata de Locusta, chaque type sexuel (mâle et femelle) et même chaque êtat physiologique (femelles en cours de maturation sexuelle et femelles mûres) peuvent être définis et individualisés par un type mitochondrial. Chez les individus du sexe mâle, l'activité des corpora allata est continue, la population mitochondriale homogène; tous les chondriosomes sont caractérisés par leur gonflement et leur grande taille, par l'éclaircissement de la substance matricielle, par le grand nombre et l'agencement régulier des crêtes.Chez les individus du sexe femelle, l'activité des corpora allata est cyclique, la population mitochondriale hétérogène. Celle-ci comprend une lignée de chondriosomes petits, à substance matricielle dense aux électrons, éléments souches permanents dans ces cellules glandulaires, et une lignée caractéristique de chaque état physiologique. Pendant la maturation sexuelle se différencie à partir de la lignée permanente une lignée de chondriosomes à substance matricielle éclaircie contenant un petit nombre de crêtes rectilignes, fines et lamellaires; certains de ces chondriosomes évoluent en mitochondries géantes caractérisées par la grande prolifération des crêtes lamellaires qui dessinent des boucles et des anneaux. En revanche, au cours des cycles ovariens s'individualisent, aux dépens de la lignée permanente, des chondriosomes très étirés qui conservent une substance matricielle dense aux électrons mais acquièrent des formes en haltère puis en cupule et participent par leur association à l'édification de volumineux complexes mitochondriaux (ehondriosphères).La signification des différents types mitochondriaux en rapport avec la forme de l'activité glandulaire et la nature du produit de sécrétion est discutée.

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Mitochondrial pleomorphism in the corpora allata of Locusta migratoria migratorioides in the course of imaginal life

Summary In the corpora allata of Locusta, each sexual type, and even each physiological state in the female reproductive cycle, are characterized by dinstinctive types of mitochondria.In males, where the activity of the corpora allata is continuous, the mitochondrial population is homogeneous; all mitochondria are large, with an electron-transparent matrix and numerous, regularly arranged cristae. In females, where the activity of the corpora allata is cyclic, the mitochondrial population is heterogeneous. It includes a line of small mitochondria with electron-dense matrix that are present throughout, and another line characteristic of specific physiological states. During sexual maturation the permanent line gives rise to a type of mitochondrion with electron-lucent matrix and a small number of cristae arranged at right angles to the long axis. Some develop into giant forms with a proliferation of cristae that form rings or loops. Furthermore, during the ovarian cycle very elongated and dumb-bell shaped mitochondria with a dense matrix make their appearance. These form voluminous complexes. The significance of the various mitochondrial types in relation to the state of glandular activity and the nature of the secretory product is discussed.