角质层的离析及显微观察方法

植物角质层的离析方法很多,获取化石植物的角质层可以同时用几种化学处理方法。舒氏液浸解是最常用的方法,也是几种方法共用时最关键的步骤。浸解液浓度过大或处理时间过长有可能破坏角质层的结构。对于已经获取到的角质层,利用相差光学显微技术可以增加反差。利用微分干涉光学显微技术不仅反差增加,而且有立体感,背景颜色也可以调节。荧光显微分析技术在不破坏标本的前提下,可以获取到表皮的特征。扫描电子显微镜具有观察不透明物体,放大范围广以及高的分辨力等优越性,可作为光学显微镜的必要补充。     

Differences in black pigmentation in lepidopteran cuticles as revealed by light and electron microscopy

Summary Black cuticles of larvae and pupae from various Lepidoptera were studied by light and electron microscopy. There are striking differences in the representation of black pigmentation, especially at the ultrastructural level. Two types may be described: 1. With the light microscope black melanin-like grana, electron-dense electron microscopically, are found in the distal parts of the exocuticle. This type is demonstrated in larvae of Celerio euphorbiae, Papilio machaon, and Phalera bucephala. 2. With the light microscope a dark homogeneous layer in the distal exocuticle can be recognized, however, electron microscopically no structures correlated with this dark pigment layer. This type of pigmentation was present in pupae of Pieris brassicae and Aglais urticae; in Pieris larvae the dark pigmented layer appeared to be limited to the epicuticle. In Celerio processes of the epidermal cells are involved in transporting precursors to the exocuticle. The conclusion was reached that black pigmentation in cuticles is based on different mechanisms as proposed by structural features. The two likely mechanisms are melanization and sclerotization.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 87, project A1, granted to Prof. Bückmann)     

The nature of driving forces for passive water transport through arthropod cuticle

1. 1.|We demonstrate using thermodynamic arguments that water loss through arthropod epicuticle is well described by a linear law relating water flux to transmembrane vapour pressure drop.

2. 2.|The relationship applies equally to systems where the liquid or vapour exist on either side of a membrane.

3. 3.|An earlier claim by some workers that water diffusion through arthropod epicuticle is proportional to chemical potential drop across the membrane is found to be theoretically unjustified.

4. 4.|Recent measurements with Periplaneta cuticle support the prediction that flux at a given temperature is proportional to the difference in vapour pressure.

Autofluorescence lifetime variation in the cuticle of the bedbug Cimex lectularius

The decay time of the fluorescence of excited molecules, called fluorescence lifetime, can provide information about the cuticle composition additionally to widely used spectral characteristics. We compared autofluorescence lifetimes of different cuticle regions in the copulatory organ of females of the bedbug, Cimex lectularius. After two-photon excitation at 720 nm, regions recently characterised as being rich in resilin showed a longer bimodal distribution of the mean autofluorescence lifetime τ_m (tau-m) at 0.4 ns and 1.0–1.5 ns, while resilin-poor sites exhibited a unimodal pattern with a peak around 0.8 ns. The mean lifetime, and particularly its second component, can be useful to distinguish resilin-rich from resilin-poor parts of the cuticle. The few existing literature data suggest that chitin is unlikely responsible for the main autofluorescent component observed in the resilin-poor areas in our study and that melanin requires further scrutiny. Autofluorescence lifetime measurements can help to characterise properties of the arthropod cuticle, especially when coupled with multiphoton excitation to allow for deeper tissue penetration.     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.     

通用引物双重PCR在节肢动物物种鉴定中的应用

【目的】本研究旨在使用基于线粒体基因通用引物的双重PCR技术同时扩增单一样本中两条标记基因,从而达到简化节肢动物物种鉴定流程的目的。【方法】在一次PCR实验中同时加入可扩增线粒体COI基因和16S rDNA两个不同分子标记的引物,对3纲8目14科的14种节肢动物物种标本的基因组DNA进行扩增;扩增产物经电泳和胶回收后测序,并BLAST在线搜索相似序列,验证基于通用引物的双重PCR在不同的动物类群中用于物种鉴定的有效性。【结果】应用基于COI和16S rDNA的引物从分属于3纲8目14科的14种节肢动物基因组DNA中均可成功扩增目的基因;扩增产物测序结果进一步证实了扩增的准确性。【结论】通过本方法进行物种的分子鉴定,不仅可以保证物种鉴定的高准确率,还可以明显减少时间与DNA样本量的消耗,这对需要快速准确鉴定物种或珍稀的材料样本十分重要。     

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.     

The adenohypophysis of the flounder,Pleuronectes flesus,and the minnow,Phoxinus phoxinus

Summary The pituitary gland of the flounder, Pleuronectes flesus, showed several unusual cytological features. Between the RPD and the PPD was a zone of cells that stained purple with Alcian blue—PAS—orange G. Many of these cells were apparently degenerating. In the PPD the strands and coils of presumptive STH cells showed a tremendous variation in both size and staining properties. In the PI there were two cell types, the PAS-positive one bordering the neurohypophysis. Around the periphery of the PI was a zone of chromophobic cells, and throughout the PI were numerous intracellular and extracellular acidophil spheres.No well defined ACTH cells were found in the RPD of the minnow (Phoxinus phoxinus). The Alcian blue—PAS—orange G technique distinguishes between blue TSH cells and purple GTH cells in the RPD and PPD. GTH cells from animals collected in the winter were vacuolated. The PI contained two cell types whose staining reactions and ultrastructure were extremely variable. Intra- and extra-cellular acidophil spheres were present.I should like to thank Dr. T. Kerr of the Zoology Department, Leeds University, for his help and encouragement, and Mr. J. Dingley of the Department of Zoology, Aberystwyth, for collecting the flounders.     

Autoradiographic and fine structural study of chitin deposition in the cuticle of a barnacle using [3H]-D-glucosamine incorporation

[³H)-D-Glucosamine was injected into the rostral sinus of Balanus eburneus (barnacle) and the distribution of labelled chitin in the cuticle was studied with autoradiography and electron microscopy. When the pattern of labelling was examined in different body regions of the same organism where thickness of fully formed cuticle varied, it was observed that the rate of chitin deposition varied, being greater in thick than in thin regions. The density of Ag grains overlying cuticle was also greater in the thick regions. When the pattern of labelling was examined in regions of cuticle, comparable in thickness, taken from a series of organisms sacrificed at different time points a comparable value for the rate of chitin deposition was obtained. In addition, asynchrony in deposition of cuticle in different body regions of the same organism as well as uptake of the label by substances other than chitin, i.e. glycogen and glycoprotcins were described.     

Ultrastructure of the Anterior Body Region of Marine Nematode Deontostoma californicum

The ultrastructure of the anterior body region of the free-living marine nematode Deontostoma calilornicum was studied by electron microscopy. The body wall consists of a nine-layered cuticle, a cellular hypodermis containing eight nerve bundles, and a well-developed coelomyarian somatic musculature. Nerves in the dorsal, lateral, ventral, and submedian hypodermal chords anterior to the nerve ring were observed with regularity. Structure of subventral somatic setae suggests a mechanoreceptive function. The esophagus is cellular and consists of three marginal cells alternating with an equal number of radial muscle cells, three esophageal glands, and three enteric nerves. The membranes of adjacent esophageal cells are sinuous. Apices of the triradiate lumen are connected with the outer wall of each marginal cell by bands of electron-dense nonmyofibrils, whereas two types of myofilaments run radially between the apophyses of the lumen and the outer walls of radial cells. Each myofibril, which forms hemidesmosomes at both ends, is interpreted to be the morphological equivalent of one sarcomere. Synaptic junctions between the processes of muscles, gland cells, and axons of the enteric nerves are described in detail.     

Comparative study of the cuticle in some aquatic oligochaetes (Annelida: Clitellata)

An examination of the cuticle of six aquatic oligochaete species using transmission electron microscopy revealed a larger morphological variation than previously known. Three freshwater species, Aulodrilus pluriseta, Spirosperma ferox (both Tubificidae), and Pristina breviseta (Naididae), and three marine species, Clitellio arenarius, Heterochaeta costata (both Tubificidae), and Paranais litoralis (Naididae), were investigated. The arrangement of the collagen fibers in the cuticle differs among the studied species. Only S. ferox shows an "orthogonal grid," i.e., layers of parallel fibers perpendicular to each other, as earlier described for lumbricids and enchytraeids. Clitellio arenarius and H. costata have fibers arranged in layers, while A. pluriseta and P. litoralis have irregularly distributed fibers. Pristina breviseta lacks cuticular fibers. The matrix surrounding the collagen fibers (when present) continues outside the fiber layer, making up a thin epicuticle, which has a unique banding in each of the studied species. The external surface of the epicuticle is covered with epicuticular projections. Their number, shape, and attachment to the epicuticle vary among the studied species. Furthermore, a distinctive internal substructure of the projections was observed in H. costata, A. pluriseta, S. ferox, and P. breviseta. Microvilli, extensions from the epidermal cells, penetrate the cuticle and terminate at its outer surface. In three species microvilli were observed to pinch off the epicuticular projections. The size, number, and shape of the latter vary; no typical microvilli were observed in S. ferox.     

Structural and ultrastructural features of boar bulbourethral glands

The morphological features of boar bulbourethral glands were examined by light and transmission microscopy. Bulbourethral glands are compound tubuloalveolar glands surrounded by a capsule of dense connective tissue and arranged in multiple lobules formed by endpieces and excretory ducts. Endpieces and excretory ducts are both lined by a single epithelium of mucous cells with a basal nucleus. Epithelial cells accumulate secretory granules containing neutral and carboxylated acid mucosubstances and a small amount of sulphated acid mucosubstances. The ultrastructure of epithelial cells varies according to the secretory cycle. In initial stages, the cells show a columnar shape and secretory granules unevenly distributed in the cytoplasm. As the synthesis of mucosubstances progresses, the amount of the secretory granules increases and the cellular shape becomes pyramidal. Secretory granules can contain inclusions and present differences among them according to their different phases of formation. In pyramidal cells, secretory products are released into the lumen by a merocrine mechanism.     

The MIXTA-like Transcription factor MYB16 is a major regulator of cuticle formation in vegetative organs

Cuticle secreted on the surface of the epidermis of aerial organs protects plants from the external environment. We recently found that Arabidopsis MIXTA-like R2R3-MYB family members MYB16 and MYB106 regulate cuticle formation in reproductive organs and trichomes. However, the artificial miRNA (amiRNA)-mediated knockdown plants showed no clear phenotypic abnormality in vegetative tissues. In this study, we used RNA interference (RNAi) targeting MYB16 to produce plants with reduced expression of both MYB16 and MYB106. The rosette leaves of RNAi plants showed more severe permeable cuticle phenotypes than the myb106 mutants expressing the MYB16 amiRNA in the previous study. The RNAi plants also showed reduced expression of cuticle biosynthesis genes LACERATA and ECERIFERUM1. By contrast, expression of a gain-of-function MYB16 construct induced over-accumulation of waxy substances on leaves. These results suggest that MYB16 functions as a major regulator of cuticle formation in vegetative organs, in addition to its effect in reproductive organs and trichomes.     

Studies on the effect of denervation in developing muscle

Summary Ultrastructural diversification of muscle fibers, with regard particularly to myofibrillar changes, has been investigated in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles of the rat during fetal and postnatal development in the presence and in the absence of motor innervation. The band pattern and the shape of the myofibrils were uniform in fetal and neonatal muscle fibers and underwent differential changes during the first weeks after birth, concomitantly with fiber type specialization. The most evident variations in myofibrillar structure arising in this period concern the thickness of the Z band and the arrangement of the myofibrils. Myofibril formation was at first not impaired by denervation of rat muscles performed in utero and, although focal disintegration of myofibrils and detachment and loss of orientation of filaments became apparent by one week, atrophic muscle fibers with well-organized myofibrils could be seen as late as 2 months after birth. However, denervated muscle fibers of EDL and soleus did not display any significant and consistent difference in myofibrillar band pattern and shape. No variation in mitochondrial content and sarcoplasmic reticulum development was likewise seen in muscle fibers of EDL and soleus after fetal denervation. The findings emphasize the importance of neuromuscular interactions in muscle differentiation.This investigation was supported in part by a grant from Muscular Dystrophy Associations of America, Inc. to Prof. M. Aloisi. A preliminary report of part of this work was presented at the XL Congress of the Italian Zoological Society, Garda, 1971 (Schiaffino, 1972).     

Ultrastructure and cytochemistry of the gastric mucosa of a reptile,Tiliqua scincoides

Summary The gastric mucosa of a reptile, the lizard Tiliqua scincoides, has been examined by light and electron microscopy. The gastric pits lead into glands that are extensively coiled in the proximal stomach but become progressively shorter and straighter in the distal stomach. The following epithelial cell types have been identified: (i) Surface mucous cells (SMC) line the entire lumenal surface as well as the pits. They contain mucus granules that stain with periodic acid-Schiff and, like the granules of mammalian SMC, commonly contain an electron dense core that appears not to be mucus (periodic acid-chromic acid-silver methenamine nonreactive). (ii) Glandular mucous cells are present in glands throughout the mucosa. They are probably homologous with the mucous neck and antral gland cells of mammals; like SMC their mucus granules contain nonglycoprotein cores. (iii) Oxynticopeptic cells (OPC) are the predominant cell type in the proximal glands but become infrequent distally. Their fine structure resembles that of OPC in other nonmammalian vertebrates, with features like those of both parietal cells and zymogen cells of mammals, (iv) Endocrine cells of three different types have been identified. Two of these show close similarities to the EC and ECL cells of mammals.The authors thank Mrs. D. Flavell for technical assistance. This study was supported by a grant from the Clive and Vera Ramaciotti Foundations     

Fine structure of isolated fibrils from the cuticle of Lumbricus sp.

A method has been developed to isolate and purify cuticular fibrils of Lumbricus. Polarizing microscopy confirms the collagenous nature of the isolated fibrils. Study in the electron microscope of isolated fibrils, negatively or positively stained, shows that they are cylindrical, unbranching and without periodic structure. Enzymatic treatment of cuticles with alpha-amylase and trypsin results in a more or less complete dissociation of the fibrils which appear clearly to be made up of helically wound bundles of filaments (30-40 A). The biophysical data and compared to the ultrastructural organization of other periodically cross-banded fibrils.     

Design of an insect cuticle associated with osmoregulation: the porous plates of chloride cells in a mayfly nymph

In mayfly nymphs of the genus Coloburiscoides, cell complexes with an osmoregulatory function (so-called chloride cells) are found in the integuments of the oral gills, the abdominal gills and gill filaments, the coxae and the thoracic sternites. The cuticle overlying each cell complex is a rigid circular plate which is known to be porous to colloidal lanthanum suspensions. The present study shows that the plate is composed only of the cuticulin and dense layers of the epicuticle. Both layers have substructures built of subunits on almost perfect hexagonal lattices. The lattice spacings are 53 and 9.5 nm for the dense layer and the cuticulin layer respectively. During moulting the apical plasma membrane of the chloride cell remains adpressed to the old porous plate. The new porous plate is formed from a new chloride cell which intrudes from the base of the integument. Throughout the moult small pores persist in the new and otherwise continuous cuticle to allow continuity of the cytoplasm of the apical and basal portions of the old chloride cell. It is thought that this phenomenon allows osmoregulatory function of the chloride cell complex to be maintained during the moult.