东亚飞蝗Locusta migratoria manilensis（Mey．）膝下器中感觉细胞的超微结构

东亚飞蝗滕下器由具橛感器组成,每一具橛感器主要由三类细胞组成,即：感觉细胞、感橛细胞和冠细胞。感觉细胞位于最近端．细胞核圆且大,细胞质内含有丰富的线粒体、高尔基体、多泡小体等细胞器。感觉细胞向近端发出轴突进入中枢神经系统,向远端发出树突。树突内古有大量的线粒体、纵行微管,树突内最复杂的结构当属纤毛根,从近端到远端依次由远端基体、近端基体、主纤毛根和纤毛小报组成。树突顶端,由远端基体发出一条感觉纤毛,纤毛具有典型的“9 0”结构。主纤毛根和纤毛小根具有明暗相间横纹结构,两横纹间的间隔距离约为65nm。感觉纤毛穿过由感橛细胞形成的感橛空隙,末端进入一高电于密度的顶端细胞外结构——帽。感橛细胞内最明显的特征为具有感橛,感橛细胞围绕着远端树突和感觉纤毛部分,冠细胞紧密地包围着感橛细胞和帽。东亚飞蝗膝下器中同时古有一或两个感觉细胞的具橛感器,这在以往研究报道中是较为少见的。本研究的主要目的在于为以后对此器官的生理功能研究提供形态学的基础材料。     

The association of proctolin with a ventral abdominal muscle of Locusta migratoria

The association of proctolin with the external ventral protractor muscle of the VIIth abdominal segment (M234) of Locusta migratoria was investigated using immunohistochemistry and RP-HPLC in conjunction with the sensitive locust oviduct bioassay. Immunohistochemistry of whole-mount tissues revealed two proctolin-like immunoreactive axons in N2B_2b1 (the nerve branch which innervates M234) as well as immunoreactive processes and varicosities on the surface of M234. Immunogold staining of M234 demonstrated that the proctolin-like immunoreactivity was present in electron-dense granules in its motor terminals. A material indistinguishable from proctolin and with proctolin-like bioactivity co-eluted with authentic proctolin on two different RP-HPLC systems. Bath application of proctolin at concentrations greater than 10^-11 M resulted in a dose-dependent increase in the neurally-evoked fast twitch amplitude and duration of M234. Concentrations greater than 10^-9 M resulted in a dose-dependent increase in basal tonus of M234. These results indicate that proctolin, or a peptide very similar to proctolin, is present in the motor innervation of M234 and acts as a cotransmitter and/or neuromodulator at this typical fast skeletal muscle.Abbreviations M234 external ventral protractor muscle of the Vllth abdominal segment - RP-HPLC reverse-phase high pressure liquid chromatography - TFA trifluoroacetic acid     

Effect of azadirachtin A on neuroendocrine activity in Locusta migratoria

Summary The neurosecretory activity of azadirachtin A-treated Locusta migratoria that failed to mature was compared with that of rapidly maturing control females by means of histological techniques. High resolution drymount autoradiography was used to localize [22,23-³H₂]dihydroazadirachtin A in the brain and corpus cardiacum (CC). The neurosecretory system of the locusts treated with this insecticide was accompanied by an unusually high accumulation of paraldehyde fuchsin (PAF)-stainable neurosecretory material (NSM) in the brain fibers and in the storage lobes of the CC. The control females never had such an intense accumulation of NSM. The accumulation of NSM, resulting from its poor release, may influence its synthesis which is controlled by feed-back regulation. [22,23-³H₂]Dihydroazadirachtin A is localized mainly at the peripheral blood-brain barrier and does not penetrate into the brain effectively, whereas it completely covers the CC. It is concluded that azadirachtin A affects the endocrine activity of the brain in an indirect way.     

Possible involvement of ecdysteroids in embryonic diapause of Locusta migratoria

In the Ibaraki population (Japan) of Locusta migratoria, adult locusts produce diapause eggs under short-day (SD) conditions and non-diapause eggs under long-day (LD) conditions. The identity and titre of ecdysteroids in the ovaries and eggs from LD and SD adult females were investigated by RIA/HPLC. Maternal ecdysteroids accumulated in the developing ovaries represented about 90% polar conjugates, 5% free ecdysteroids and 5% non-hydrolyzable metabolites. Before oviposition the quantity of ecdysteroids reached 29.8±1.85 ng 20-hydroxyecdysone equiv. per mg tissue ovaries from LD females and 13.1±3.55 ng 20E equiv./mg in ovaries from SD females. The sum of RIA-positive materials in newly laid eggs was more than three times higher in non-diapause eggs than in diapause eggs. Ecdysteroids present in egg extracts comprised about 85% polar conjugates, 5% free ecdysteroids and 10% non-hydrolyzable metabolites. On the other hand, after diapause termination the amount of ecdysteroids increased drastically. Also, the composition of ecdysteroids differed from that observed during diapause and became comparable to that of non-diapause eggs. The significant differences in the ecdysteroids between non-diapause and diapause eggs may suggest the possible involvement of these compounds in the control of embryonic diapause of this locust.     

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.     

Effects of stimulation and rest on the ultrastructure of the excitatory neuromuscular junctions of Locusta migratoria L.

Summary The terminals of the fast axon on extensor tibiae muscle fibres of Locusta were examined in untreated nerve-muscle preparations and in preparations stimulated electrically at frequencies varying from 0.5 to 100 Hz. The ultrastructure of the terminals in preparations stimulated at the lower range of these frequencies, which induce twitch contractions of the muscles, is similar to that of the controls. Stimulation at the higher frequencies induced tetanic muscle responses and rapid fatigue of the muscles after which they would not respond again to high frequency stimulation for about 1 h. This loss and recovery of the responses of the muscles is correlated with changes in the ultrastructural appearance of the terminals, in particular in the number and shape of the synaptic vesicles. The ultrastructure of these recovering axon terminals closely resembles that of the controls.R.P. Botham gratefully acknowledges the SRC for financial assistance     

The innervation of the corpus cardiacum of Locusta migratoria: A neuroanatomical study with the use of Lucifer yellow

Summary Neural connections of the corpus cardiacum (CC) in the African locust, Locusta migratoria, were labelled with the fluorescent tracer Lucifer yellow. (1) Unilateral anterograde labelling of the nervus corporis cardiaci I revealed fluorescent fibres in the storage lobe of the CC (CCS). Some fluorescent fibres in the CCS closely approached the ipsilateral border of the glandular lobes of the CC (CCG). Fluorescent fibres also projected into the neuropile of the hypocerebral ganglion via the ipsilateral nervi cardiostomatogastrici I and II, and from there into the oesophageal nerves. (2) Unilateral anterograde labelling of the nervus corporis cardiaci II revealed fluorescent fibres in the CCS and in the ipsilateral CCG. Fluorescent fibres also projected via the ipsilateral nervus corporis allati I into the corpus allatum. (3) Unilateral retrograde labelling of the nervus corporis allati I revealed a distinct fluorescent nerve tract that runs through the CCS and into the nervus corporis cardiaci II. The tract arises from about eight cell bodies in the brain at the rostroventral side of the ipsilateral calyx of the mushroom body. (4) Labelling of the recurrent nerve revealed fluorescent fibres and some fluorescent cell bodies in the hypocerebral ganglion and, via the nervi cardiostomatogastrici I and II, also in the CCS. Fluorescent fibres were also present in the oesophageal nerves.     

Immunocytochemical demonstration of octopamine-immunoreactive cells in the nervous system of Locusta migratoria and Schistocerca gregaria

Summary The distribution of octopamine in the metathoracic ganglion, brain and corpus cardiacum of Locusta migratoria and Schistocerca gregaria was investigated by means of immunocytochemistry with an antiserum against octopamine. The dorsal unpaired median (DUM) cells of the metathoracic ganglion were found to be strongly octopamine-immunoreactive. In the rostroventral part of the protocerebrum a group of seven immunopositive cells was demonstrated. Stained nerve fibres of these cells run into three directions: circumoesophageal connectives, midbrain, and optic lobes. As far as the protocerebrum is concerned, immunoreactive fibres were found in the central body, the protocerebral bridge, and in other neuropile areas. In the optic lobe a dense plexus of immunopositive fibres was found in the lobula and in the medulla. In the brain one other immunopositive cell was demonstrated, situated at the lateral border of the tritocerebrum. Octopamine could not be shown to occur either in the globuli cells of the mushroom bodies or in the dorsolateral part of the protocerebrum, where the perikarya of the secretomotor neurones are located that innervate the glandular cells of the corpus cardiacum. In the nervi corporis cardiaci II, which contain the axons of the neurones that extend into the glandular part of the corpus cardiacum, and in the corpus cardiacum proper no specific octopamine immunoreactivity could be found.     

Changes in chemoreceptor sensitivity in relation to dietary selection by adult Locusta migratoria

During somatic growth in adult Locusta migratoria L. there are variations in the responsiveness of chemosensilla on the maxillary palps. Relative changes in chemosensitivity to amino acids and sucrose are consistent with relative changes in protein and carbohydrate ingestion. There is a decline throughout the somatic growth phase in the proportion of sensilla responding to stimulation. In those sensilla which respond, there is a decline in the firing rate to amino acids, while sucrose responsiveness declines to a lesser degree. We suggest that variations in the levels of nutrients in the storage tissues, especially the haemolymph, arising from changes in the demand for protein and carbohydrate for growth, directly or indirectly, influence gustatory sensitivity.

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Résumé Pendant la croissance de L. migratoria L. les réactions aux acides aminés et aux sucres des sensilles chimiques des palpes maxillaires changent. Ces changements relatifs de la sensibilité chimique à ces deux aliments correspondent aux modifications dans l'ingestion de protéines et d'hydrates de carbone par cet insecte. La diminution de la réponse aux acides aminés est liée à la fois au nombre de sensilles qui réagissent et à leur taux de réaction. La sensibilité des sensilles au sucrose varie moins profondément et implique uniquement des modifications du nombre de sensilles réagissant. Nous suggérons que des variations de la teneur en éléments nutritifs de l'haemolymphe, dues à des modifications de la demande en protéines et en glucides nécessaires à la croissance, influent sur la sensibilité gustative.

     

Ultrastructural differentiations in the developing follicle cortex of Locusta migratoria,with special reference to vitelline membrane formation

Summary Electron microscopic studies on developing follicles of Locusta migratoria show the vitelline membrane to be composed of two ultrastructurally distinguishable components: The vitelline membrane bodies (VMBs) and, in addition, fine granular material, cementing the VMBs together. VMBs form first in the oocyte-near zone within the oocyte-follicle cell space. Subsequently, the second vitelline membrane substance is secreted between the VMBs through apical protrusions of the follicle cells. The possible origin of the VMBs is discussed.Yolk uptake in Locusta seems to occur predominantly by pinocytosis. During oocyte development the oocyte membrane is enlarged by numerous microvilli and folds. In addition pinocytotic vesicles are pinched off. It is supposed that the latter loose their coat and eventually transform into large proteid yolk spheres.This work was supported by the Volkswagenstiftung, HannoverI wish to thank Prof. Dr. H. Emmerich, Techn. Hochschule Darmstadt, for valuable discussions     

Ultrastructural and immunocytochemical studies of neuromuscular junctions in oviduct of Locusta migratoria

The ultrastructure of nerve endings in the oviduct visceral muscles of Locusta migratoria was studied by electron microscopy and by immunogold labeling for two kinds of neuromodulators, the pentapeptide proctolin and FMRFamide-related peptides. Nerve endings contained electron-lucent round vesicles and two kinds of granules (round and avoid), and formed two types of synapses or release sites with the muscle. The morphologically distinct nerve endings were classified into three different categories based on the composition of synaptic vesicles and granules. Type-I nerve endings were dominated by electron-lucent round vesicles and contained only a few round electron-dense granules. Type-II nerve endings contained mostly electron-dense round granules and electron-lucent round vesicles. A few electron-dense ovoid granules were also present. Electron-dense ovoid granules dominated the type-III nerve endings, which usually contained less electron-lucent vesicles than either type-I or II nerve endings. Both proctolin and FMRFamide-like immunoreactivity was associated with electron-dense round granules. However, FMRFamide-like immunoreactivity was only found in the type-II nerve endings, while proctolin immunoreactivity was found within type-I nerve endings as well as in some type-II nerve endings. Immunological results therefore allow us to further divide type-II nerve endings into type-IIa (immunonegative for proctolin) and type-IIb (immunopositive for proctolin). Type-III nerve endings show no immunolabeling to either proctolin or FMRFamide.     

Crustacean cardioactive peptide-immunoreactive neurons innervating brain neuropils,retrocerebral complex and stomatogastric nervous system of the locust,Locusta migratoria

The distribution and morphology of crustacean cardioactive peptide-immunoreactive neurons in the brain of the locust Locusta migratoria has been determined. Of more than 500 immunoreactive neurons in total, about 380 are interneurons in the optic lobes. These neurons invade several layers of the medulla and distal parts of the lobula. In addition, a small group of neurons projects into the accessory medulla, the lamina, and to several areas in the median protocerebrum. In the midbrain, 12 groups or individual neurons have been reconstructed. Four groups innervate areas of the superior lateral and ventral lateral protocerebrum and the lateral horn. Two cell groups have bilateral arborizations anterior and posterior to the central body or in the superior median protocerebrum. Ramifications in subunits of the central body and in the lateral and the median accessory lobes arise from four additional cell groups. Two local interneurons innervate the antennal lobe. A tritocerebral cell projects contralaterally into the frontal ganglion and appears to give rise to fibers in the recurrent nerve, and in the hypocerebral and ingluvial ganglia. Varicose fibers in the nervi corporis cardiaci III and the corpora cardiaca, and terminals on pharyngeal dilator muscles arise from two subesophageal neurons. Some of the locust neurons closely resemble immunopositive neurons in a beetle and a moth. Our results suggest that the peptide may be (1) a modulatory substance produced by many brain interneurons, and (2) a neurohormone released from subesophageal neurosecretory cells.     

Ultrastructural changes in the glial cells at neuromuscular synapses of Locusta migratoria occurring after nerve stimulation and subsequent rest

The glial processes ensheathing the motor nerve terminals on the retractor unguis muscle of Locusta migratoria are described. Ultrastructural changes observed after electrical nerve stimulation (20 Hz, 7 min) without or with subsequent rest (2 min, 1 h) are analysed morphometrically. Immediately after stimulation both the average terminal circumference (+ 23%) and its proportion covered by glial processes (+ 16%) are significantly increased. The mean number of Schwann cell processes per micron of terminal circumference (without stimulation: 0.86 +/- 0.04) is also affected: Immediately after stimulation it is increased by about 15% and after 2 min of rest even by 36%. The periaxonal cleft (without stimulation: 16.5 nm +/- 0.36) becomes wider immediately after stimulation by about 19%, an effect which is almost reversed after 1 h of rest. It is suggested that these changes are a consequence of the enlargement of the nerve terminal's surface upon massive exocytotic activity and that they are possibly mediated by mechanical attachment between glial and terminal plasma membranes.     

GABA-like immunoreactivity of identified interneurons in the flight system of the locust,Locusta migratoria

Summary The transmitter content of identified inhibitory interneurons in the flight system of the locust, Locusta migratoria, has been characterized using antibodies raised against protein-conjugated gamma aminobutyric acid. Identified flight neurons were filled with the fluorescent dye, Lucifer Yellow. Serial sections of dye-filled neurons were incubated with an antibody to gamma aminobutyric acid which was subsequently tagged with a fluorescent marker. Excitatory motoneurons to wing muscles and 13 flight interneurons (3 excitatory, 7 inhibitory, and 3 with unknown synaptic effect) were examined. Neither the moto-neurons nor any of the 3 excitatory interneurons contained immunoreactive material. Six of the 7 inhibitory interneurons did contain immunoreactive material. All the neurons which contained immunoreactive material and whose synaptic effect is known were inhibitory. We conclude that most of the inhibitory flight interneurons which have been described use gamma aminobutyric acid as their transmitter. Interestingly, at least 1 set of interneurons known to be inhibitory does not use gamma aminobutyric acid. We predict that the 2 interneurons which do contain immunoreactive material and whose synaptic effect is not yet known will be found to have inhibitory roles in the operation of the flight circuitry.     

Elektronenmikroskopische Untersuchungen des ventralen Diaphragmas von Locusta migratoria und der langsamen Kontraktionswelle nach Fixation durch Gefriersubstitution

Zusammenfassung Die Muskulatur des ventralen Diaphragmas von Locusta besteht aus parallel verlaufenden Muskelfasern, die über glanzscheibenartige Strukturen in den Querverbindungen miteinander verbunden sind. Jede Muskelfaser ist von einer dicken Bindegewebshülle umgeben. Die Fasern sind über Hypodermiszellen mittels Tonofibrillen in der Cuticula verankert.Unkontrahierte Sarkomere haben eine Länge von 5 m und mehr. Eine H-Zone ist angedeutet, eine M-Linie nicht vorhanden. Aktin- und Myosinfilamente (Durchmesser 72 bzw. 160 AE) liegen nicht im Register. Daneben existiert ein dritter, sehr dünner Filamenttyp. Die Z-Zone hat einen gewellten Verlauf und faßt die Aktinfilamente in Bündeln zusammen. Mitochondrien liegen beiderseits der Z-Zone. Das T-System faltet sich in Form von Sarkolemmkerben in das Faserinnere ein und setzt sich in Tubuli nach innen fort. Z-Zonen und Sarkolemmkerben sind miteinander verbunden. T-System und sarkoplasmatisches Retikulum treten durch unregelmäßig verteilte Diaden miteinander in Kontakt.Begrenzter Ca⁺⁺-Entzug läßt Kontraktionswellen von der Länge mehrerer Sarkomere entstehen.Die Fixation durch Gefriersubstitution erzeugt gegenüber Standardverfahren Veränderungen wie Schrumpfung des Sarkoplasmas, Verdickung der Myosinfilamente, Vakuolisierung von Mitochondrien und vesikulärem System. In der Kontraktionswelle verkürzt sich die A-Zone mit zunehmender Sarkomerenkontraktion. Der Durchmesser der Myosinfilamente beträgt 172 AE, die Periodik der Cross-bridges von 305 AE bleibt im mittleren Bereich der Filamente konstant.

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Electron microscope studies of the ventral diaphragm of Locusta migratoria and of the slow wave of contraction after fixation by freeze substitution

Summary The muscular system of the ventral diaphragm of Locusta consists of parallel muscle fibers, which are connected by structures like intercalated discs within transverse bridges. Each muscle fiber is enveloped by a thick sheath of connective tissue. The fibers are attached to the cuticule by means of hypodermic cells with tonofibrils.Uncontracted sarcomeres have a length of 5 m and more. The H-band is slightly indicated, a M-line is not visible. Actin and myosin filaments (diameter 72 respectively 160 AE) are out of register. Moreover there is a third and very thin type of filaments. The Z-band has an undulating shape and collects the actin filaments into bundles. Mitochondria lie on either side of the Z-band. The T-system invaginates as sarcolemmal clefts and continues its course inwards as tubuli. The sarcolemmal clefts are connected with the Z-band. The T-system and the sarcoplasmic reticulum are joined by diads of irregular distribution.Limitated deprivation of Ca⁺⁺ causes waves of contraction with the length of several sarcomeres.Contrary to standard methods the freeze-substitution causes some modifications such as shrinking of the sarcoplasm, thickening of the myosin filaments, vacuolization of mitochondria and vesicular system. Within the waves of contraction the A-band shortenes with increasing sarcomere contraction. The diameter of the myosin filaments measures 172 AE, the 305 AE-period of the cross-bridges remains constant within the middle of the filaments.

Auszug aus der Dissertationsarbeit Elektronenmikroskopische Untersuchungen der Muskulatur des ventralen Diaphragmas von Locusta migratoria und der langsamen Kontraktionswelle unter Anwendung der Gefriersubstitution (Mathematisch-Naturwissenschaftliche Fakultät der Universität Göttingen). Auf Anregung von Herrn Prof. Dr. Schlote und mit Unterstützung durch die Deutsche Forschungsgemeinschaft und die Göttinger Akademie der Wissenschaften.     

Ageing adipokinetic cells in Locusta migratoria: an ultrastructural morphometric study

Summary A morphometric study was made of the ultrastructure of adipokinetic cells in resting adults of Locusta migratoria at 3, 23, and 43 days after imaginal ecdysis. The nucleus, rough endoplasmic reticulum, and Golgi apparatus enlarge with age, which indicates that the synthesis and packaging of secretory substances increases during ageing. The size of the storage compartment, consisting of secretory and ergastoplasmic granules, does not increase earlier than 23–43 days after imaginal ecdysis. The lysosomal compartment markedly enlarges between 3 and 23 days; later on, the growth of this compartment, especially of autophagosomes, is less prominent. This suggests that lysosomal destruction initially compensates for the production of new secretory granules, assuming that exocytosis of secretory granules by adipokinetic cells is insignificant in resting locusts. Afterwards, lysosomal destruction may no longer be sufficient to prevent over-production of secretory granules, as is suggested by the increase in the number of these granules between 23 and 43 days. This coincides with the appearance of a considerable number of large ergastoplasmic granules, which represent a spatially more efficient form of storage of secretory material than the much smaller secretory granules. The increase with age in the amount of secretory products indicates that the biosynthetic activity of the adipokinetic cells is not (finely) tuned to their releasing activity.     

Serotonin-immunoreactive neurones in the brain of Locusta migratoria innervating the corpus cardiacum

Summary The serotoninergic innervation of the corpus cardiacum (CC) of Locusta migratoria was investigated using two antisera against serotonin. A dense network of immunoreactive nerve fibres was present in the storage lobe of the CC. Immunopositive fibres only sporadically crossed the border between the storage lobe and the glandular lobe of the CC. Immunopositive fibres entered the storage lobe of the CC via the nervus corporis cardiaci I (NCCI); NCCII was immunonegative. Unilateral retrograde fillings of the NCCI with the fluorescent tracer Lucifer yellow, followed by antiserotonin immunocytochemistry, revealed about 20 double-labelled neurones in the anterior part of the pars intercerebralis. The double-labelled neurones were scattered between fluorescent non-immunoreactive neurones. Additionally, 5–7 neurones labelled only with Lucifer yellow were found at the ventrolateral side of the tritocerebrum. No immunopositive neurones were observed in the hypocerebral ganglion. Immunopositive fibres from neurones in the frontal ganglion ran via the recurrent nerve and the neuropile of the hypocerebral ganglion into the paired oesophageal nerve. At most, a few immunopositive nerve fibres occurred in the cardiostomatogastric nerves II, which connect the storage lobe of the CC with the paired oesophageal nerve at the caudal end of the hypocerebral ganglion.