CUTICLE DEVELOPMENT ON GLANDULAR TRICHOMES OF CANNABIS SATIVA (CANNABACEAE)

The dermal sheath of glandular trichomes of Cannabis sativa L., consisting of cuticle and a subcuticular wall, was examined by transmission electron microscopy. Cuticle thickened selectively on the outer wall of disc cells of each trichome prior to formation of the secretory cavity, whereas thickening was less evident on the dermal cells of the bract. Membraned secretory vesicles that differ in size and appearance in the secretory cavity were the source of precursors for synthesis of cuticle. Vesicle contents, released following the degradation of the vesicle membrane upon contact with the subcuticular wall, contributed to both structured and amorphous phases of cuticle development. The structured phase was represented by deposition and thickening of cuticle at the subcuticular wall-cuticle interface to form a thickened cuticle. In the amorphous phase precursors permeated the cuticle in a liquid state, as shown by fusion of cuticles and wax layers between contiguous glands, and may have contributed to growth in surface area of the expanding sheath. Disc cells are interpreted to control growth of secretory cavity by secretion of membraned vesicles into the cavity. The thickened cuticle, which increased eightfold in thickness during enlargement of the gland, provided structural strength for the extensive surface area of the dermal sheath. The gland of Cannabis in which vesicle contents contribute to the growth in thickness and surface area of the cuticle of the sheath is interpreted to represent a phylogenetically derived state as contrasted to secretory glands possessing only cuticle and lacking a complement of secretory vesicles.     

Tomato fruit continues growing while ripening,affecting cuticle properties and cracking

Fruit cuticle composition and their mechanical performance have a special role during ripening because internal pressure is no longer sustained by the degraded cell walls of the pericarp but is directly transmitted to epidermis and cuticle which could eventually crack. We have studied fruit growth, cuticle modifications and its biomechanics, and fruit cracking in tomato; tomato has been considered a model system for studying fleshy fruit growth and ripening. Tomato fruit cracking is a major disorder that causes severe economic losses and, in cherry tomato, crack appearance is limited to the ripening process. As environmental conditions play a crucial role in fruit growing, ripening and cracking, we grow two cherry tomato cultivars in four conditions of radiation and relative humidity (RH). High RH and low radiation decreased the amount of cuticle and cuticle components accumulated. No effect of RH in cuticle biomechanics was detected. However, cracked fruits had a significantly less deformable (lower maximum strain) cuticle than non‐cracked fruits. A significant and continuous fruit growth from mature green to overripe has been detected with special displacement sensors. This growth rate varied among genotypes, with cracking‐sensitive genotypes showing higher growth rates than cracking‐resistant ones. Environmental conditions modified this growth rate during ripening, with higher growing rates under high RH and radiation. These conditions corresponded to those that favored fruit cracking. Fruit growth rate during ripening, probably sustained by an internal turgor pressure, is a key parameter in fruit cracking, because fruits that ripened detached from the vine did not crack.     

Helical disposition of proteins and lipopolysaccharide in the outer membrane of Escherichia coli

In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.     

Tissue origins,cell lineages and patterns of cell division in the developing dermal system of the frut of Vitis vinifera L.

Cell division during development of the dermal system of fruit of the grape cv. Gordo is confined to the first growth period. The epidermis is conserved with anticlinal proliferative cell divisions providing for increase in cell number. The hypodermis is the layer of origin of the collenchymatous dermal system. Six or seven layers are differentiated by periclinal cell divisions early in the first growth period, and later increase in size is obtained by proliferative anticlinal cell divisions. These observations are related to developmental and genetic control of fruit shape and volume.     

Ultrastructure of the Pharyngeal Cuticle and Lectin Labelling with Wheat Germ Agglutinin-gold Conjugate Indicating Chitin in the Pharyngeal Cuticle of Oesophagostomum dentatum(Strongylida, Nematoda)

The ultrastructure of the pharynx of the first juvenile stage and of adult Oesophagostomum dentatum (Nematoda, Strongylida) is investigated by TEM. Additional light microscopical and SEM data are presented for the anterior alimentary canal of all life-history stages and for adult O. quadrispinulatum . Ultrathin sections of adult and the first juvenile stage O. dentatum are labelled with the lectin wheat germ agglutinin coupled to colloidal gold. The pharyngeal cuticle consists of a trilaminate epicuticle and an amorphous basal layer, which also exhibits individual fibres in adult worms. The pharyngeal cells of the first juvenile stage are already arranged as in the adult worm. In comparison with data for additional nematodes taken from the literature, a trilaminate epicuticle and an amorphous basal layer are postulated for the pharynx in the ground pattern of the Nematoda. Lectin-gold labelling reveals a strong binding to the pharyngeal cuticle of O. dentatum . Competitive inhibition experiments with N -acetyl-D-glucosamine and triacetylchitotriose, as well as treatment with chitinase, neuraminidase and sulphuric acid, indicate that the pharyngeal cuticle contains chitin. This finding is supported by staining of the pharyngeal cuticle by the fluorescent brightener calcofluor white in cryosections of O. dentatum . The occurrence of chitin is interpreted as an original character of the Nematoda. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Development of respiratory structures in embryos and first and second instars of the bark scorpion,Centruroides gracilis (Scorpiones: Buthidae)

The SEM was used to study the development of respiratory structures in successive stages in relation to the overall changes occurring in the scorpions. Book lung development is a slow process, starting with spiracles and a sac‐like atrium in the early embryo and continuing lamellar formation to 150 or more in the adult. In the embryo, the primordial epithelial cells become aligned in a planar pattern as they secrete granules of material that aggregate spontaneously to form the cuticular walls of the lamellae. A blade‐like structure is formed consisting of cells sandwiched within the two cuticle walls they secreted. These cells are in the primordial air channel. The adjacent hemolymph channel is nearly devoid of cells, but cross‐bridges develop and help stabilize the cuticle walls and maintain the width of the channel. The cells in the primordial air channel undergo cytolysis, leaving it open for air except for cuticular cross‐bridges. Development continues in the newborn (first instars); the air channels of some lamellae still contain cells and are not yet functional for gas exchange. The first instars are weak and relatively inactive. They climb up on the mother's dorsum until the first molt (about 8 days). With the cuticular walls of the lamellae in place, cells adhering to the wall in the hemolymph channel produce a thin, new tissue layer (epithelium) on the lamellar wall facing the hemolymph channel. This layer has many discontinuities as though it is slowly developing. Formation of the tissue layer and cytolysis of the cells in the air channels continue through the first molt in which little book lung cuticle is shed as exuvium. The air channels of the second instars (foraging nymphs) are now cell free and open for air passage except for the cross‐bridges. The tissue layer is still incomplete and continues to be formed. It may provide the hypodermal primordium for cuticle replacement in later molts, but development was not studied beyond the second instar except for comparison with book lungs in the adult. The blade‐like lamellae in the adult are larger and more numerous than in the second instar, but in the anterior book lung the shape of the cuticle wall and cross‐bridges and the widths of the air and hemolymph channels are about the same as in the second instar. The air channels in the posterior part of the lamellae have distinctive, vein‐like space‐holders. The similarity of the adult anterior lamellae with those in the second instar suggests retention of this part through the 4–5 molts to maturation, and/or cell processes like those in the embryo are repeated, but this needs to be examined in further studies of cell and cuticle changes before and during the molts. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

龙舌草果实和种子及其种苗发育形态学研究

采用形态观察和GMA切片相结合的方法,对龙舌草果实、种子及种苗发育的形态结构进行解剖学观察研究,探讨龙舌草个体发育中表现出的与水生环境相适应的结构特征.结果显示:(1)龙舌草由佛焰苞包被的果实通常由6心皮构成,心皮边缘不完全愈合,层片状胎座,水面形成的幼嫩果实被果柄拉入水中发育成熟;种子具毛,萌发时苗端先于根端发育,在胚根分化之前先出现单细胞的下胚轴毛,随后胚根发育为初生根,并在真叶的节处发育出不定根;果皮和叶肉内的维管束仅由少数细胞构成,且细胞分化不明显,有发达的通气道;叶表皮无角质层,细胞外凸,叶肉细胞数量少;表明龙舌草的结构特征与水生环境相适应.(2)研究还发现,龙舌草的种子无胚乳,下胚轴贮存种苗发育需要的大量淀粉粒,以及初生根和不定根均有根毛的结构,且与泽泻科的慈菇和泽泻的结构一致,证明水鳖科与泽泻科具有较近的亲缘关系.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

An overview on plant cuticle biomechanics

Plant biomechanics combines the principles of physics, chemistry and engineering to answer questions about plant growth, development and interaction with the environment. The epidermal-growth-control theory, postulated in 1867 and verified in 2007, states that epidermal cells determine the rate of organ elongation since they are under tension, while inner tissues are under compression. The lipid cuticle layer is deposited on the surface of outer epidermal cell walls and modifies the chemical and mechanical nature of these cell walls. Thus, the plant cuticle plays a key role in plant interaction with the environment and in controlling organ expansion. Rheological analyses indicate that the cuticle is a mostly viscoelastic and strain-hardening material that stiffens the comparatively more elastic epidermal cell walls. Cuticle stiffness can be attributed to polysaccharides and flavonoids present in the cuticle whereas a cutin matrix is mainly responsible for its extensibility. Environmental conditions such as temperature and relative humidity have a plasticizing effect on the mechanical properties of cuticle since they lower cuticle stiffness and strength.The external appearance of agricultural commodities, especially fruits, is of great economic value. Mechanical properties of the cuticle can have a positive or negative effect on disorders like fruit cracking, fungal pathogen penetration and pest infestation. Cuticle rheology has significant variability within a species and thus can be subjected to selection in order to breed cultivars resistant to pests, infestation and disorders.     

Effect of the new fluorescent brightener Rylux BSU on morphology and biosynthesis of cell walls in Saccharomyces cerevisiae

Rylux BSU, a new fluorescent brightener from the family of 4,4-diaminostilbene-2,2disulfonic acid derivatives, inhibited growth and cytokinesis of the yeast Saccharomyces cerevisiae. In the presence of 0.1–1 mg/ml Rylux BSU the cells grew in clumps, had irregular shape and were larger than controls. They formed apparently normal primary septa but their secondary septa and lateral cell walls, especially those in older cells, were abnormally thick with large deposits of amorphous wall material in the periplasmic spaces all over the cell surface. Chitin content in the cell walls of cells grown in the presence of Rylux BSU was increased 2 to 5 times in comparison to that of the controls and glucan content was reduced by up to 30%. In the in vitro assays with particulate membrane fractions, Rylux BSU acted as a non-competitive inhibitor of -1,3-glucan synthase with inhibitory constant K _i=1.75 mg/ml whereas the chitin synthase was inhibited to a much lesser extent. From the difference of the effects of Rylux BSU on the synthesis of chitin in vivo and in vitro it is concluded that the brightener interacts with chitin synthase only indirectly, possibly by influencing the properties of integral plasma membrane.Abbreviations RBSU Rylux BSU, 1,4-benzenedisulfonic acid-2,2-[ethyleneidy]bis[(3-sulpho-4,1-phenylene)imino[6-bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diylamino]]bis-, hexasodium salt - FB fluorescent brightener     

Immunochemical localization of tetrahydrocannabinol (THC) in cryofixed glandular trichomes of Cannabis (Cannabaceae)

Delta 9-tetrahydrocannabinol (THC) localization in glandular trichomes and bracteal tissues of Cannabis, prepared by high pressure cryofixation-cryosubstitution, was examined with a monoclonal antibody-colloidal gold probe by electron microscopy (EM). The antibody detected THC in the outer wall of disc cells during the presecretory cavity phase of gland development. Upon formation of the secretory cavity, the immunolabel detected THC in the disc cell wall facing the cavity as well as the subcuticular wall and cuticle throughout development of the secretory cavity. THC was detected in the fibrillar matrix associated with the disc cell and with this matrix in the secretory cavity. The antibody identified THC on the surface of secretory vesicles, but not in the secretory vesicles. Gold label also was localized in the anticlinal walls between adjacent disc cells and in the wall of dermal and mesophyll cells of the bract. Grains were absent or detected only occasionally in the cytoplasm of disc or other cells of the bract. No THC was detected in controls. These results indicate THC to be a natural product secreted particularly from disc cells and accumulated in the cell wall, the fibrillar matrix and surface feature of vesicles in the secretory cavity, the subcuticular wall, and the cuticle of glandular trichomes. THC, among other chemicals, accumulated in the cuticle may serve as a plant recognition signal to other organisms in the environment.     

Use of a Fluorescent Brightener to Demonstrate Cellulose in the Cellular Slime Molds

The presence and location of cellulose in different stages of the life cycles of the cellular slime molds can be demonstrated by use of the disodium salt of 4,4'-bis(4-anilino-6-bis (2-hydroxyethyl)-amino-s-triazin-2-ylamino)-2,2' -stilbene disulfonic acid, a fluorescent brightener. It may be used successfully as a direct stain at a concentration of 0.1% in half-normal saline at pH 6; and it may be incorporated into growth media as a vital stain at a concentration of 0.0025% with no inhibitory effect at any developmental stage. Vegetative myxamoebae contain no cellulose and show no fluorescence in the presence of this brightener when viewed with ultraviolet light. In later stages of the life cycle, the time and sites of cellulose formation can be demonstrated with the brightener because of its fluorescence. e.g., in the slime covering of the pseudoplasmodia, in the sorophore sheath, in the walls of stalk cells and spores, in the walls of microcysts, and in the walls and sheath material of macrocysts. The brightener appears to be a very sensitive indicator for cellulose, and it has certain advantages over other cellulose stains, since the staining reaction (fluorescence) is very intense, long-lasting, and not obscured by unstained cellulose-free myxamoebae if such are present.     

Light- and electron-microscopical studies on the structure of salt glands of Tamarix aphylla L.

Cuttings of Tamarix aphylla were grown in various concentrations of NaCl. Salt glands on newly developed branches were sectioned and examined, both with the light- and with the electron-microscope. An electron-dense material appears in cuttings grown in all concentrations of NaCl at the anticlinal walls of the innermost pair of secretory cells and, to some extent, on the surface of the gland above the cuticle. In the glands of cuttings grown in high concentration of NaCl this material also appears in large quantities, not only on top of the gland but also beneath the cuticle at the region of the median anticlinal wall of the outermost pair of secretory cells. The material beneath the cuticle is connected to the electron-dense material on the outer surface of the cuticle via the secretory pores. It is suggested that this material is of pectic nature and forms a continuous system with the walls and wall protuberances of the secretory cells which are also very rich in pectic substances. As pectic substances absorb solutions easily they may enhance the transport of salt.
Many proplastids, vacuoles with simple structure and others with infolded membranes as well as numerous mitochondria were observed in the secretory cells. In the walls between the collecting cells and the innermost secretory cells there are numerous plasmodesmata with characteristically arranged membranes.     

Local motion and conformational changes in the cuticle of Clivia miniata Regel

The temperature dependence of the local diffusion of fluorescent molecular probes of various polarities (alkane, long-chain fatty acid, short-chain alcohol and fatty acid), all labelled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl in the cuticle of Clivia miniata Regel was studied by the technique of fluorescence recovery after photobleaching. The technique yields the coefficient of diffusion, D, in the plane of the cuticle over distances of some 10 m and the fraction, R, of mobile reporter molecules. The inner (more hydrophilic) and the outer (more hydrophobic) faces of the cuticle were studied separately by appropriate incubation. The value of D was found to depend sensitively on the polarity of the probe, the temperature and the position within the cuticle (outer hydrophobic or inner hydrophilic side). Depending on the type of probe, D increased (in the case of the alkane) or decreased (in the case of the alcohol) after removal of the (monomeric) waxes. The electron-spin-resonance (ESR) spectra of incorporated spin-labelled fatty-acid probes measured in the intact cuticle contained a major component similar to the spectrum recorded from the polymerized matrix from which waxes had been extracted, and a second component similar to the spectrum from the monomeric waxes. At low temperatures, the ESR spectra from labels at two different chain positions corresponded to chain motion which was slow on the ESR timescale. At high temperatures, the spectral component from the monomeric waxes indicated chain motions in the motional narrowing regime which were of an essentially isotropic nature.No evidence was found for a liquid-crystalline lipid phase such as found for the polar lipids in cell membranes, nor was there evidence for a sharp, thermotropic, lipid-phase transition either in the cuticle or in the waxes. Experiments with oriented samples did not demonstrate the presence of large domains with a uniform orientation of the lipid chains relative to the cuticular layers. The diffusion measurements and spin-label studies provide evidence for conformational changes of the cuticle extending over the whole temperature range studied (10° C to 70° C). These conformational changes are attributed to phase-separation processes within the cuticle. The phase separation in extracted waxes extended over a similar broad temperature range. This indicates that the transitions in the cuticle are largely determined by these components. At higher temperatures, however, the chain mobility in the regions of monomeric wax was considerably greater than that in the polymerized matrix. The experimental results strongly indicate that all three layers of the Clivia cuticle exhibit a multilamellar structure of alternatingly stacked, highly hydrophobic layers of welldefined thickness (5±0.5 nm) and more disordered layers of variable (4 to 15 nm) thickness. The lamellae are wellordered and extend over the whole leaf in the cuticle proper but are split-up into small domains in the inner and the external cuticular layer. Furthermore, changes of the molecular transport properties caused by the influence of ozone exerted during the growth of the plant were studied. We found that the diffusion coefficient increased both in the outer and the inner layer of the cuticle. A particularly large increase, by about a factor of three, was found for alkane diffusion in the hydrophobic outer face, pointing to defects in the polymerized matrix.Abbreviations MX-membrane polymer matrix membrane (or monomeric wax-depleted cuticle) - ESR electron-spin resonance - n-SASL n-(4,4-dimethyl-N-oxy-2-oxazolidinyl)-stearic acid - NBD 7-nitrobenz-2-oxa-1,3-diazol-4-yl The present work was made possible by a grant from the Bayerische Umweltministerium. Additional support by the Fonds der Chemischen Industrie is gratefully acknowledged. We are most grateful for very helpful discussions with Professor H. Ziegler, Professor J. Schönherr and Dr. M. Riederer from the Institut für Botanik, Technische Universität München, FRG.     

Morphogenesis of the antenna of the male silkmoth. Antheraea polyphemus, III. Development of olfactory sensilla and the properties of hair-forming cells

During adult development of the male silkmoth Antheraea polyphemus, the anlagen of olfactory sensilla arise within the first 2 days post-apolysis in the antennal epidermis (stage 1-3). Approximately on the second day, the primary dendrites as well as the axons grow out from the sensory neurons (stage 4). The trichogen cells start to grow apical processes approximately on the third day, and these hair-forming 'sprouts' reach their definite length around the ninth day (stages 5-6). Then the secretion of cuticle begins, the cuticulin layer having formed on day 10 (stage 7a). The primary dendrites are shed, the inner dendritic segments as well as the thecogen cells retract from the prospective hair bases, and the inner tormogen cells degenerate around days 10/11 (stage 7b). The hair shafts of the basiconic sensilla are completed around days 12/13 (stage 7c), and those of the trichoid sensilla around days 14/15 (stage 7d). The trichogen sprouts retract from the hairs after having finished cuticle formation, and the outer dendritic segments grow out into the hairs: in the basiconic sensilla directly through, and in the trichoid sensilla alongside, the sprouts. The trichogen sprouts contain numerous parallel-running microtubules. Besides their cytoskeletal function, these are most probably involved in the transport of membrane vesicles. During the phase of cuticle deposition, large numbers of vesicles are transported anterogradely from the cell bodies into the sprouts, where they fuse with the apical cell membrane and release their electron-dense contents (most probably cuticle precursors) to the outside. As the cuticle grows in thickness, the surface area of the sprouts is reduced by endocytosis of coated vesicles. When finally the sprouts retract from the completed hairs, the number of endocytotic vesicles is further increased and numerous membrane cisterns seem to be transported retrogradely along the microtubules to the cell bodies. Here the membrane material will most probably be used again in the formation of the sensillum lymph cavities. Thus, the trichogen cells are characterized by an intensive membrane recycling. The sensillum lymph cavities develop between days 16-20 (stage 8), mainly via apical invaginations of the trichogen cells. The imago emerges on day 21.     

Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.     

The Pneumathodes of Laguncularia racemosa: Little Known Rootlets of Surprising Structure, and Notes on a New Fluorescent Dye for Lipophilic Substances

Abstract: Two types of negatively geotropic aerial roots may be observed on the root system of Laguncularia racemosa: pneumatophores with secondary growth, and short-lived pneumathodes which remain in the primary anatomical state. The pneumathodes distinguish themselves by the absence of an epidermis; instead, the outer cortex takes the place of the outermost tissue. This tissue forms a three-dimensional network of rod-like cells and gas spaces. The cell walls contain a lipophilic substance which ensures that the intercellular spaces remain gas-filled during submergence. An uniseriate cellular layer separates the outer and inner cortex. This uniseriate cellular layer, which we term a "pore layer", is characterized by cells with suberized and lignified cell walls and occasional pores among the cells. The pores permit the diffusion of oxygen-rich air from the surface of the pneumathode to the aerenchyma of the inner cortex and the escape of carbon dioxide from the interior of the root. The structure of the differentiated pneumathode originates from frequent cell divisions in the part of the apical meristem where the outer cortex emerges. Because of the pressure thereby exerted on the epidermis and hypodermis, these two cell layers tear and become separated from the outer cortex. Their remnants remain visible at the base of the pneumathode and as an appendage of the calyptra. The function and significance of the pneumathodes for L. racemosa are discussed. An extract of Xanthoria parietina was employed as a new fluorescent dye to stain suberine in cell walls. The staining technique is presented in this paper.     

Fine structure of the epicuticle of the desert scorpion, Hadrurus arizonensis, with reference to location of lipids.

The surface and transverse sections of the epicuticle of the desert scorpion, Hadrurus arizonensis, were examined by scanning and transmission electron microscopy, respectively. Sclerite cuticle that was untreated prior to normal EM preparative procedures was compared to cuticle subjected to lipid solvents, high temperature, and concentrated alkali. Surface morphology of untreated intersegmental cuticle was also examined. The epicuticle is composed of four sublayers: outer membrane, outer epicuticle, cuticulin, and the dense homogeneous layer. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax canals that penetrate the cuticulin layer even though the solvents effectively remove lipids from the epicuticle for chemical analysis. The surface of the sclerite cuticle contains amorphous particles, crystalline projections, and scattered openings to dermal gland ducts. Perforations that correspond to the opening of wax canals were faintly visible after extraction of surface waxes and clearly visible after KOH treatment. No openings to dermal gland ducts or wax canals were observed in untreated intersegmental cuticle. However, wax canals are likely obscured by surface waxes similar to those present in sclerite cuticle.     

On the pronymphal stage in the migratory locust (Locusta migratoria, Orthoptera, Acrididae)

The structure of the integument, somatic and visceral muscles, midgut, and Malpighian tubules were investigated at the late stages of the embryonic and early postembryonic development of the migratory locust, Locusta migratoria, to assess the organization of its pronymphal stage. In its morphogenetic features, the vermiform locust larva sometimes called the pronymph corresponds to the first nymphal instar covered with the second embryonic cuticle which has not been shed. Since the first-instar locust nymphs before and after the shedding of this embryonic cuticle differ significantly in many morphological characters, two consecutive phases of this nymphal instar can be distinguished: the first phase existing from the moment of development of the third embryonic cuticle to the shedding of the second one; the second phase existing from the shedding of the second embryonic cuticle to the formation of the cuticle of the second nymphal instar. Since the pronymphal stage should precede the nymph stage, it may be concluded that the pronymph of the locust is fully embryonized and covered with the second embryonic cuticle, which is also typical of other insects with hemimetabolous development (Konopová and Zrzavý, 2005). Therefore, it would be erroneous to refer to the vermiform first-instar nymph as the pronymph, because the two stages are separated by molting and formation of a new cuticle.     

Fine structure of the cuticle of the black widow spider with reference to surface lipids

The surface and transverse sections of the cephalothorax, abdomen, and walking leg cuticle of the black widow spider, Latrodectus hesperus, were examined by scanning and transmission electron microscopy. Cuticle that was untreated prior to normal EM preparative procedures was compared with cuticle subjected to lipid solvents and/or concentrated alkali. The surface of untreated dorsal cephalothorax cuticle contained droplets and a lipid film that obscured fine surface detail. Immersing the cuticle in chloroform: methanol removed the droplets and lipid film, exposing previously covered openings to dermal gland ducts. An epicuticle, exocuticle, and endocuticle were present in all transverse sections of cuticle as was a complex system of pore and wax canals that connected the epidermis with the cuticle surface. The epicuticle of the walking leg was composed of three sublayers: outer membrane, outer epicuticle, and the dense homogeneous layer. A cuticulin layer was not observed. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax/pore canals.