Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by electron-microscopic gold-labelling immunocytochemistry

An immunogold-labelling electron-microscopic study of the frontal ganglion of two noctuids, Lacanobia oleracea and Helicoverpa armigera, has been carried out with antisera directed against three neuropeptides; allatostatins of the Y/FXFGL-NH₂ type, Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin. The ganglion of both noctuids has two pairs of large peptidergic neurones with many clusters of electron-dense granules, one pair being situated anteriorly and the other posteriorly. By means of a double-labelling (flip-flop) technique, with different sizes of gold particles, all possible paired combinations of the three different types of peptide have been visualised within granules of the anterior neurones, leading to the conclusion that the three peptides are co-packaged and co-stored in these cells. Within the posterior neurones of L. oleracea, gold labelling of granules is only linked to the Y/FXFGL-NH₂ allatostatin antisera and, in contrast to the anterior cells of this species in which double gold labelling results in a sparse accumulation of gold particles for any one peptide type, single labelling gives a more intense, uniform pattern of gold particles. In contrast to L. oleracea, the gold-labelling pattern seen in the posterior neurones of H. armigera reflects the co-localisation of allatostatins of the Y/FXFGL-NH₂ type with Mas-AS in this species. Allatotropin is absent in the posterior neurones of both species.Grant funding was from the Wellcome Trust: grant no. 068105 (A.T.)     

Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2)

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose-response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.     

中国东南沿海8种蛸的齿舌形态学研究

近几十年来,依赖于物种表型特征的捕述[1-3],有关头足类种质鉴定和分类体系构建等方面的研究工作不断得到完善和补充[4-6].与分子遗传学标记不同,形态特征受环境因子(如水温、盐度、底质等)影响,而且这些变异的遗传组成又很难确定.     

Morphological studies of different mitochondrial populations in monkey myocardial cells

Summary The ultrastructure of mitochondria in monkey myocardial cells was investigated by scanning electron microscopy, thin sections and freeze-fracturing. Mitochondria with well-developed cristae were distributed around the nucleus, between the myofibrils and beneath the sarcolemma. Those clustered near the the poles of the nucleus were generally spherical in shape. Interfibrillar mitochondia were arranged in longitudinal rows between the myofibrils, were elongated and usually about the same length as a sarcomere. Subsarcolemmal mitochondria varied in size and shape, being rod-like, spherical, polygonal or horseshoe-like. There were usually two profiles of subsarcolemmal mitochondria in each section of sarcomere, although sometimes one or three occurred, and they were typically oriented perpendicularly to the myofibrils. These morphological differences among mitochondria could reflect functional and/or mechanical properties in the various cellular locations.     

Isolation and characterization of temperature-sensitivelethal(2)giant larva alleles

Summary In this paper we present an analysis of the behavior ofl(2)gl ^tsimaginal wing discs during culture in adult hosts. Thel(2)gl ^tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl ^tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl ^tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl ^tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl ^tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl ^tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl ^tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl ^tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl ⁺function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.     

Pigment movements in fish melanophores: Morphological and physiological studies

Summary The ultrastructure of the melanophores of Pterophyllum scalare was studied with respect to changes in cell shape during melanosome migration and the number and distribution of microtubules within the cell extensions. Cells were fixed with pigment fully aggregated or fully dispersed. All measurements were carried out on cross sections of cell processes, i.e. sections cut perpendicular to the long axis of the cell extensions. Cross sections of processes of melanophores with dispersed pigment are more or less ovoid in shape, and microtubules are arranged predominantly just below the cell membrane. These microtubules exhibit a relatively constant centre-to-centre spacing of about 55–65 nm. Processes of melanophores with aggregated pigment seem to be collapsed; their volume is substantially decreased but their circumference equals that of dispersed melanophores. The number of microtubules is reduced, and their regular arrangement is lost. The differences in microtubule number associated with the aggregated or dispersed state occur irrespective of the nature of the agent inducing dispersion or aggregation. In addition, apparent insertion of microtubules into the plasma membrane of the cell processes and associations of microtubules with cytoplasmic densities in the cell centre are described.The results indicate a rapid disassembly and assembly of microtubules associated with pigment movements. The possible role of microtubule associations with cell membrane and densities as sites of microtubule polymerization is briefly discussed.This work was supported by a grant from the Deutsche Forschungsgemeinschaft.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

20-Hydroxyecdysone-induced differentiation and apoptosis in the Drosophila cell line, l(2)mbn

20-Hydroxyecdysone has an inhibitory effect on the proliferation of l(2)mbn cells, causes vacuolization and fragmentation of cells, and promotes a strong phagocytotic activity. From several lines of evidence, it can be concluded that 20-hydroxyecdysone induces apoptosis. Long-term video observations following the fate of individual cells, scanning and transmission electron microscopy reveal the typical characteristics of apoptosis: sequestration of small cellular protuberances or larger parts of the cell with nuclear fragments (apoptotic bodies), chromatin condensation, condensation and vesiculation of cytoplasm, whereas the mitochondria retain their normal appearance. The induction of apoptosis by 20-hydroxyecdysone was confirmed by the TUNEL reaction and quantitatively determined by a method based on this reaction. Onset of apoptosis precedes phagocytotic activity. JH III alone has no clear-cut effect on l(2)mbn cells. In double treatments, the inhibitory effect of 20-hydroxyecdysone on cell proliferation is significantly reduced by the addition of JH III. Whether or not JH III also reduces apoptotic activity is not yet clear. It is shown that the l(2)mbn cell line is an advantageous model system for the exploration of steroid-induced apoptosis.     

The metathoracic wing-hinge chordotonal organ of an atympanate moth,Actias luna (Lepidoptera,Saturniidae): a light- and electron-microscopic study

Summary The structure of a simple chordotonal organ, the presumed homologue of the noctuoid moth tympanal organ, is described in the atympanate moth, Actias luna. The organ consists of a proximal scolopidial region and a distal strand, which attaches peripherally to the membranous cuticle ventral to the hindwing alula. The strand is composed of elongate, microtubule-rich cells encased in an extracellular connective tissue sheath. The scolopidial region houses three mononematic, monodynal scolopidia, each comprised of a sensory cell, scolopale cell, and attachment cell. The dendritic apex is octagonally shaped in transverse section, its inner membrane lined by a laminated structure reminiscent of the noctuoid tympanal organ collar. A 9+0-type cilium emerges from the dendritic apex, passes through both the scolopale lumen and cap, and terminates in an extracellular space distal to the latter. Proximal extensions of the attachment cell and distal prolongations of the scolopale cell surrounding the cap are joined by an elaborate desmosome, with which is associated an extensive electron-dense fibrillar plaque. Within the scolopale cell, this plaque constitutes the scolopale rod material. The data are discussed in terms of both the organ's potential function, and its significance as the evolutionary proto-type of the noctuoid moth ear.     

Evolution and ultrastructure of the bovine spermatogonia precursor cell line

We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra-and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophateinsensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with malespecific pheromone-sensitive afferents in the macro-glomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echo-thiophate-insensitive AChE activity. These neurous seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antenno-cerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.Abbreviations ACh acetylcholine - AChE acetylcholinesterase - AL antennal lobe - AMMC antennal mechanosensory and motor center - ChAT choline acetyltransferase - IACT inner antenno-cerebral tract - MGC macroglomerular complex     

Plasma-membrane glycoproteins during spermiogenesis and in the spermatozoa of some Orthoptera

Summary Orthoptera spermatids and spermatozoa from two species of Tettigoniidae and from one of Acrididae were analysed by means of the fluorescent lectins, concanavalin A or wheat-germ agglutinin, with the aim of finding -D mannose· -D glucose· N-acetylglucosamine and sialic acid sugar residues in their plasmamembrane glycoproteins. Labelling with lectins shows remarkable changes occurring in the plasma membrane during spermiogenesis. In early spermatids, the whole cell surface is labelled, but in mature spermatids and spermatozoa, a noticeable fluorescence is restricted to the membrane that covers the acrosome. The end piece of the tail of Acrididae spermatids and spermatozoa fluoresces after wheat-germ agglutinin labelling. The intense labelling of acrosomal area is independent of acrosomal size and shape, as shown by the marked differences observed in the acrosomes of Tettigoniidae compared with Acrididae: in the former, the acrosome is a well-developed structure with an arrow-like shape, but in Acrididae, the acrosome resembles a small vesicle in the anterior tip of the cell. The large amount of some sugar residues in the plasma membrane covering the acrosome is discussed in relation to the features observed in other species, and also in connection with the physiology of the male gamete prior or during fertilization.     

Effects of cold-exposure and subsequent recovery on cellular proliferation with influence of 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2)

In developing insects, the peak level of 20-hydroxyecdysone (20E) initiates a decrease in cyclin expression, which subsequently triggers an arrest of cellular proliferation and the start of differentiation, finally culminating in the moult. We investigated the impact of cold-exposure (4 °C) and recovery (26 °C) on the cell cycle activity of the Plodia interpunctella Lepidoptera cell line IAL-PID2 and on the expression of B-type cyclin (PcycB), ecdysone receptor (B1-isoform; PiEcR-B1), and Hsc70 (PiHsc70) mRNA. Cold-exposure significantly reduced expression of these mRNAs, while their levels increased to above control values during subsequent recovery at the normal growth temperature. When cold-exposed cells were returned to 26 °C, cell cycle activity restarted, but apoptosis was strongly increased. The presence of 20E appeared to increase this apoptotic phenomenon. This result is consistent with the described protective role of 20E against a variety of stressors and with the capacity of 20E to induce cell death in different situations. Here, we illustrate for the first time a connection between 20E treatment and Hsc70 expression during cold-exposure and subsequent recovery in insect cells. Combined with the 20E-induced apoptotic response, our results suggest that regulation of Hsc70 expression by 20E could act in synergy with the control of apoptotic cell death in order to optimize the survival of specific cell populations after a period of cold-exposure.     

Corrosion casts as a method for investigation of the insect tracheal system

Summary The tracheal systems of five insect species (two species of ants, worker bee, housefly and the cabbage butterfly) have been studied by scanning electron microscopy of corrosion casts. This technique, which is commonly used for the investigation of vertebrate vasculature, is adapted to demonstrate the ultrastructure of the insect respiratory organ. The problem of filling a blind ending system was solved by injecting the resin Mercox into the evacuated tracheae through a thoracal spiracle. After polymerization of the resin, the tissue was digested enzymatically and chemically. The three-dimensional structure of the tracheal system was investigated by scanning electron microscopy. The technique used here displays for the first time the complex morphology of the entire tracheal system in fine detail, especially the structure of spiracles, airsacs, tracheae and tracheoles. Smooth-walled terminal tracheoles show up in flight muscles. The finest tracheoles that could be identified have diameters of approximately 70 nm. This approaches the finest tracheoles portrayed by transmission electron micrographs.     

Allatostatin-like immunoreactivity in the abdomen of the locust Schistocerca gregaria

A polyclonal antibody against allatostatin 1 (AST-1) of cockroach Diploptera punctata was used to investigate the distribution of AST-like immunoreactivity within the abdomen of the locust, Schistocerca gregaria. In the abdominal ganglia, AST-like immunoreactivity was found in both cell bodies and neuropile. In ganglia 6 and 7, staining was found in serial homologous cell bodies in anterior dorsolateral and dorsomedial, and posterior ventrolateral and ventromedial locations. In the terminal ganglion, the numerous immunoreactive somata were smaller in size than those in the unfused ganglia. The combination of backfill experiments with immunocytochemistry showed that, in abdominal ganglion 7, one neuron of the ventromedian cell body cluster and two of the ventrolateral cluster innervated the oviduct, which itself was covered with a dense mesh of AST-immunoreactive varicosities. Combining electron microscopy with immunohistochemistry revealed AST-like immunoreactivity in dense-core vesicles located in neurohaemal-like terminals lacking structures normally found in synapses. A mesh of AST-immunoreactive varicosities was also found on the muscles of the spermatheca and the spermathecal duct. In addition, a mesh of strongly stained varicosities was present in the distal perisympathetic organs (neurohaemal organs in the abdomen) and on the lateral heart nerves (a putative neurohaemal release zone). These data indicate that AST is an important neuroactive substance that is probably involved in multiple tasks in the control of the locust abdomen.     

Morphological specializations of dorsal rim ommatidia in the compound eye of dragonflies and damselfies (Odonata)

We have examined the fine structure of dorsal rim ommatidia in the compound eye of the three odonate species Sympetrum striolatum, Aeshna cyanea and Ischnura elegans. These ommatidia exhibit several specializations: (1) the rhabdoms are very short, (2) there is no rhabdomeric twist, and (3) the rhabdoms contain only two, orthogonally-arranged microvillar orientations. The dorsal rim ommatidia of several other insect species are known to be anatomically specialized in a similar way and to be responsible for polarization vision. We suggest that the dorsal rim area of the odonate compound eye plays a similar role in polarization vision. Since the Odonata are a primitive group of insects, the use of polarized skylight for navigation may have developed early in insect phylogeny.     

In vitro peptidergic neurons from the adult locust pars intercerebralis: Morphological and immunocytological studies

Primary cell cultures were prepared from a major neurosecretory center of the adult locust brain, the pars intercerebralis, in order to characterize neurosecretory cells growingin vitro. Individual pars intercerebralis could be removed free of surrounding tissue and dissociated by mechanical treatment. Mature neurosecretory neurons of different sizes regenerate new neurites during the initial three daysin vitro in serum-free medium. They show a tendency to sprout one primary neurite from which fine processes develop. By means of electron microscopy, we observed the integrity of the cellular organelles, indicating that cultured neurons are healthy, and we were able to distinguish three types of neurosecretory neurons on the basis of the ultrastructural aspects of the neurosecretory material. These three types have the same ultrastructural characteristics asin situ neuroparsin, ovary maturing parsin and locust insulin related peptide neurons. Immunogold labelling at the electron microscopic level, using the two available specific antibodies, anti-neuroparsin and anti-ovary maturing parsin, confirms the morphological characterization of neuroparsin and ovary maturing parsin cells. These results show for the first time that cultured locust neurosecretory neurons behave like thosein vivo, in terms of their ultrastructure and immunocytochemistry. Moreover, the presence of recently-formed neurosecretory material both in the Golgi zone of the perikaryon and in the neuronal processes indicates that cultured neurons have functional capacity since they are able to synthesizede novo and to transport the neurosecretory material along the neurite. Thus our well-characterized culture system provides a suitable invitro model to investigate the secretory mechanism of locust neurosecretory neurons.     

Occurrence of the genus Ophicrania Kaup (Insecta: Phasmatodea) in Panay island (Philippines) and description of a new species

A distinctive new species of the phasmatodean genus Ophicrania Kaup, 1871 (Phasmatidae: Platycraninae) is described and figured from the Philippines. Ophicrania conlei n. sp. (from Mount Madja-as, on Panay island) is characterized by the bicoloured anal region of the male hind wing, divided into an inner whitish patch, and an outer brownish area. The species is also distinguished from its most similar congeners on the basis of integumental coloration, features of the antennae, thoracic nota, wings, legs, and terminalia. The present study also provides an emended diagnosis of Ophicrania, and an updated checklist of the taxa of Platycraninae recorded from the Philippine archipelago.