Cuticle Preparations Research Articles

Mongolitria: A new Early Cretaceous three-valved seed from Northeast Asia.

Fossil seeds recovered from the Early Cretaceous of Mongolia and Inner Mongolia, China, are described and assigned to Mongolitria gen. nov., a new genus of gymnosperm seed. Abundant lignitized seeds along with compression specimens isolated from the matrix were studied using a combination of scanning electron microscopy, anatomical sectioning, light microscopy, synchrotron radiation X-ray microtomography, and cuticle preparations. A single permineralized seed was examined by light microscopy of cellulose acetate peels and X-ray microtomography. Two species are recognized, Mongolitria friisae sp. nov. and Mongolitria exesum sp. nov. Both seeds are orthotropous with a short apical micropyle and a small, basal, circular attachment scar. The thick sclerenchymatous integument has a consistently three-parted organization and about 20 conspicuous longitudinal ribs on the surface. Mongolitria exesum differs from M. friisae primarily in its much larger size and thicker seed coat, which also preserves clear evidence of insect damage. Mongolitria is similar to other fossil seeds that have been assigned to Cycadales, but displays a unique combination of characters not found in any living or extinct cycadaceous plant, leaving its higher-level systematic affinities uncertain. Germination apparently involved splitting of the integument into three valves. Mongolitria was prominent among the plant parts accumulating in peat swamps in eastern Asia during the Early Cretaceous.

A modified, step-by-step procedure for the gentle bleaching of delicate fossil leaf cuticles

Cuticular analysis has long been used by palaeobotanists for the identification of fossil leaves, and a variety of chemical procedures has been developed to extract and prepare fossil cuticles. However, even commonly used solutions may be too harsh for the preparation of extremely delicate cuticles. Here we offer a step-by-step protocol for the preparation of fragile conifer cuticles using sodium hypochlorite, otherwise known as household bleach. Conifer needles from the Miocene lignites of the Adendorf and Hambach open-mine pits in western Germany were prepared using a mild solution of this oxidizing agent. The cuticles had proven to be too fragile for most maceration chemicals, including Schulze’s reagent, which even disintegrated the cuticles that were given a protective coating. However, it was discovered that trimming the leaf margins and damaged areas prior to a short exposure to 5–10% sodium hypochlorite solution resulted in the good preparation of the cuticle. Furthermore, this modified method allowed for the preparation of large areas of leaf. While this procedure may not be suitable for all cuticles, it is offered here as an easy and gentle method for preparing extremely delicate conifer cuticles that are destroyed by other chemicals and protocols.

Practicing logical reasoning through Drosophila segmentation gene mutants.

Laboratory practical sessions are critical to scientific training in biology but usually fail to promote logical and hypothesis-driven reasoning and rely heavily on the teacher's instructions. This paper describes a 2-day laboratory practicum in which students prepare and analyze larval cuticle preparations of Drosophila segmentation gene mutant strains. Embryonic segmentation involves three major classes of genes according to their loss-of-function phenotypes: the establishment of broad regions by gap genes, the specification of the segments by the pair-rule genes, and the compartments within segments by the segment polarity genes. Students are asked to sort undefined segmentation mutants into gap, pair-rule, or segment polarity categories based on their knowledge of the Drosophila segmentation process and the microscopic anatomical traits they are capable of finding in the sample preparations. This technically simple practicum prompts students to pay attention to detailed observation to detect anatomic markers of intrasegmental compartments and thorax versus abdomen cuticle, and promote their logical reasoning in hypothesizing to which segmentation type a given mutant fits best.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, <em>Dermestes maculatus</em>

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Methods of maceration and microscopical analysis on cuticle

Cuticles are usually very resistant and can be isolated from the fossil specimens. Several methods of maceration can be applied in preparation of fossil cuticles. Maceration in Schulze’s reagent is the most common procedure and it is the most critical step in the methods. The cuticles may be destroyed by strong maceration solution with longer time. The use of microscopy of phase-contrast is very useful for very thin cuticles as it shows greater contrast.The use of differential-interference is better than use of microscopy of phase-contrast for these thin cuticles, for it enhances the relief and makes fine structures visible. Fluorescence microscopy has the advantage that no maceration procedure is necessary and so the specimens are not damaged. Scanning electron microscopy is an essential and supplementary method in the analysis of cuticles for it produces high resolution, scientifically informative, three-dimensional images.

Ultrastructure of plant leaf cuticles in relation to sample preparation as observed by transmission electron microscopy.

The leaf cuticular ultrastructure of some plant species has been examined by transmission electron microscopy (TEM) in only few studies. Attending to the different cuticle layers and inner structure, plant cuticles have been grouped into six general morphological types. With the aim of critically examining the effect of cuticle isolation and preparation for TEM analysis on cuticular ultrastructure, adaxial leaf cuticles of blue-gum eucalypt, grey poplar, and European pear were assessed, following a membrane science approach. The embedding and staining protocols affected the ultrastructure of the cuticles analysed. The solubility parameter, surface tension, and contact angles with water of pure Spurr's and LR-White resins were within a similar range. Differences were however estimated for resin : solvent mixtures, since Spurr's resin is combined with acetone and LR-White resin is mixed with ethanol. Given the composite hydrophilic and lipophilic nature of plant cuticles, the particular TEM tissue embedding and staining procedures employed may affect sample ultrastructure and the interpretation of the results in physicochemical and biological terms. It is concluded that tissue preparation procedures may be optimised to facilitate the observation of the micro- and nanostructure of cuticular layers and components with different degrees of polarity and hydrophobicity.

Preparation of Cuticles from Unhatched First-Instar Drosophila Larvae

The finely sculpted cuticle of Drosophila carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. Studies of the Drosophila cuticle have focused mainly on first-instar larvae and adult cuticular morphology. This protocol describes the preparation of cuticles from larvae that have not yet hatched from the egg. It is designed for sampling all eggs laid by one or more females. This can be particularly useful, for example, when a mutation produces embryos that are unable to hatch from the egg.

More avast! For dorsal closure: Characterization of acal and its relation with Drosophila Jun N-terminal kinase signaling

Jun N-terminal kinase (JNK) signaling regulates epithelial remodeling throughout metazoa. Drosophila melanogaster possesses only one homolog of JNK, Basket, which coordinates dorsal closure during embryonic development. During dorsal closure the lateral epidermis changes shape and stretches over the amnioserosa towards the midline, wrapping up the embryo dorsally. Mutant embryos for basket and other members of the pathway die due to failures in closure, leaving dorsal and anterior holes in cuticle preparations. In this work, we describe an uncharacterized gene whose mutants show dorsal closure defects in cuticle preparations. We also describe its relationship with Basket signaling. The locus, acal, is conserved among drosophilids at the nucleotide level, but the transcripts only have small open reading frames that are not conserved. We hypothesize they are non-coding RNA genes. acal is enriched in the nuclear fraction, and is expressed in the epidermis during dorsal closure. By genetic interactions with basket mutants, we observed that it negatively regulates Basket signaling. This was confirmed by expression of puckered-lacZ, a reporter for Basket signaling activity. Finally, to get further insight into acal, we performed genetic interactions with known inhibitors of Basket signaling. From these assays, we believe that Acal acts in conjunction with Raw and independently of Hindsight, both of which inhibit Basket signaling in the epidermis and in the amnioserosa, respectively. Work is now focused on determining the molecular function of acal and its mechanism of inhibition.

A new transfer technique to extract and process thin and fragmented fossil cuticle using polyester overlays

Extraction and chemical processing of fragmented cuticles from fossil leaves are facilitated by a new non-destructive technique. Transfer of fossil cuticle onto polyester overlays renders whole-leaf maceration unnecessary. This technique is completely safe and extremely easy to apply for the preparation of fragile and fragmented cuticles, while preserving the fossil specimen and reducing chemical processing time.

The stomatal apparatus of Lycopodium japonicum and its bearing on the stomata of the Devonian lycophyte Drepanophycus spinaeformis

The structure of the stomatal apparatus of the leaf of Lycopodium japonicum Thumb was studied using epidermal macerations, sections and scanning electron microscopy. The stomatal apparatus of L. japonicum consists of two large guard cells and pore, and is anomocytic. Based on light microscopy, the impression from epidermal macerations that there were two small guard cells surrounded by two, large, similarly shaped, subsidiary cells (paracytic) derives from a pronounced elliptical cuticular ledge on the surface of the guard cells surrounding a thickened circumporal area. A similar appearance is characteristic of cuticle preparations of the Devonian lycophyte Drepanophycus spinaeformis Goppert. We therefore conclude, as did W.H. Lang over 70 years ago, that the stomata of the early lycophyte were also anomocytic, as were those of a second species of Drepanophycus, D. qujingensis.

How Paleozoic Vines and Lianas Got off the Ground: On Scrambling and Climbing Carboniferous–Early Permian Pteridosperms

Late Paleozoic pteridosperms displayed various growth habits, including arborescent, leaning, and scrambling and/or climbing forms. This article reviews information gathered to date on vine- and liana-like forms among these plants, based on impression/compression material and cuticle preparations from the Upper Carboniferous and Lower Permian of Europe and North America. Vine- and liana-like pteridosperms used various modes of attachment for both anchorage and support. Such adaptations are very similar (and perhaps analogous) to those that exist in extant angiosperms and include hooks, leaflet tendrils, tendrils terminating in adhesive pads, and aerial adventitious roots. A number of morphological features of scrambling/climbing pteridosperms (e.g., tiny, deeply sunken stomata, marginal water pits, various types of secretory structures, and heterophylly) are considered as they relate to the autecological significance where they may be related to special physiological requirements necessary in the scrambling/climbing growth habit. We hypothesize that scrambling and/or climbing pteridosperms may have played an important role in some of the late Paleozoic coal-swamp forest ecosystems, perhaps even comparable to the role of angiospermous vines/lianas in tropical and subtropical forest ecosystems today.

Fate mapping of the larval silk glands of Bombyx mori by UV laser irradiation of the egg at fertilisation.

Localised UV laser irradiation of Bombyx eggs at the fertilisation stage causes localised cuticle defects in the integument of the resultant larvae. Based on the correlation between the site of irradiation and the site of defects, I previously established a fate map of the larval integument of Bombyx mori. Although most of the internal organs of the irradiated specimens were eliminated in the acidic fixative used for cuticle preparation, silk proteins which had accumulated in the gland lumen were resistant to the acid treatment and silk gland morphology was thus preserved. Taking advantage of this phenomenon, I continued fate mapping, now of the larval silk glands of Bombyx mori, utilising the same specimens that were prepared and analysed in the previous study. Of 710 irradiated specimens analysed, 452 showed two normal glands, 27 showed one normal and one deformed gland, 21 showed one normal gland only, and 210 showed no silk gland. The sites of irradiation that caused the deletion of a silk gland were located within a limited area that entirely overlapped the ventral half of the area where irradiation caused defects in the labium. These results are consistent with previous histological descriptions stating that the silk glands develop from the paired invaginations in the labial segment. Based on the data, the presumptive region for the larval silk glands was located on the previously established Bombyx fate map.

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster.

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do.

Transport of model peptides across Ascaris suum cuticle

Several FMRFamide-related peptides (FaRPs) found in nematodes exert potent excitatory or inhibitory effects on the somatic musculature of Ascaris suum and other nematode species when injected into the pseudocoelom or applied directly to isolated neuromuscular preparations. These peptides, however, generally fail to induce detectable effects on the neuromusculature when applied externally to intact nematodes. The apparent lack of activity for these peptides when administered externally in whole-organism assays is likely a function of both absorption and metabolism. To delineate the factors that govern transport of peptides across the cuticle/hypodermis complex of nematodes, we measured the rates of absorption of a series of structurally related model peptides using isolated cuticle/hypodermis segments from A. suum and two-chamber diffusion cells. [ 14C]-Labeled peptides were prepared from d-phenylalanine, with the amide nitrogens sequentially methylated to give AcfNH 2, Acf 3NH 2, Acf(NMef) 2NH 2 and Ac(NMef) 3NHMe. These model peptides were designed to allow systematic analysis of the influence of peptide size, hydrogen bonding and lipophilicity on transport. Results of these studies show that, within this series, permeability across the cuticle increases with addition of each methyl group. The permeability coefficient of Ac(NMef) 3NHMe, with four methyl groups, was 10-fold greater than that of the smaller peptide, AcfNH 2, even though both peptides contain five hydrogen bonds. When compared with vertebrate membranes, transport of the model peptides across A. suum cuticle was about 10-fold slower. A biophysical model for transcuticular transport of peptides predicted that nematode FaRPs, which are larger, less methylated and less lipophilic than the model peptides tested, would not be absorbed across the cuticle of nematodes. This prediction was confirmed for the excitatory FaRP, AF2 (KHEYLRFamide), which did not diffuse across the cuticle/hypodermis complex, but diffused rapidly across lipid-extracted cuticle preparations.

Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants.

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. Here we show that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp.

Molecular lesions associated with alleles of decapentaplegic identify residues necessary for TGF-beta/BMP cell signaling in Drosophila melanogaster.

We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-beta homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-beta superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embryonic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-beta-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.

Ultrastructure and chemical composition of some isolated plant cuticles in relation to their permeability to the herbicide, diuron

SUMMARYThe ultrastructure of leaf and fruit cuticles from nine species was investigated before and after their enzymatic isolation. No major alteration was observed in the internal ultrastructure of cuticles after the isolation process. The isolated cuticle preparations showed several levels of complexity from the Pyrus communis L. leaf cuticle represented by a simple layer, to the Vanilla planifolia Andr. leaf cuticle preparation having extensive cell walls on its inner face. Two main ultrastructural types were distinguished; cuticles characterized by an almost entirely reticulate structure and cuticles characterized by an outer lamellate region with an inner reticulate region. The permeability of isolated cuticles to diuron was measured with a double water compartment device using 14C‐diuron. The permeability coefficient (P) ranged from 1.9 to 40.1 nm s−1 for seven species. The mean value of P for cuticles having an entirely reticulate structure was higher than that for cuticles with an outer lamellate zone. Moreover, for each of the two ultrastructural types, the permeability decreased with an increase in soluble wax content expressed per surface unit.

CUTICLES FROM CHONDRUS CRISPUS (RHODOPHYTA)1

ABSTRACTA simple, nondestructive physical process was developed for routinely isolating the outermost layers from female, male, and sporophyte fronds of Chondrus crispus Stack‐house. Yields of pure cuticles from apical segments ranged from 0.74 to 2.35% on a dry weight basis after 5–7 d of culture. These undegraded cuticles were examined by electron microscopy (scanning and transmission electron microscopy), spectroscopy (infrared and X‐ray), and chemical means.Cuticles isolated from female or male fronds were characterized by parallel arrays of electron‐dense lamellae (typically 6–14) alternating with more electron‐transparent regions. The thickness and uniformity of these lamellae provide the physical basis for the iridescence characteristic of C. crispus fronds. Sporophyte fronds are not iridescent. This phenomenon may be explained by the fewer electron‐dense cuticular lamellae (usually three to seven) and the fact that these lamellae anastomose freely to form a thin cuticle with a highly irregular substructure. Elements detected by X‐ray analysis, in addition to carbon and oxygen, included Mg, Br, S, and Ca in both gametophyte and sporophyte cuticles. Major features of FTIR spectra of all cuticles were absorbances due to proteins. A strong band, indicative of sulfate ester, occurred near 1250 cm−1 in all cuticle preparations. Gametophyte, but not sporophyte, cuticles absorbed at 935, 846, and 800 cm−1 consistent with the presence of kappa and/or iota carrageenan. Amino acid analyses showed that sporophyte and gametophyte cuticles were generally similar in gross composition. All contained proline as the principal residue together with significant amounts of cysteine, methionine, and lysine. Protein contents calculated from these analyses ranged from 37.6 to 44.4% on a dry weight basis as compared to 51.5–56.7% calculated from total nitrogen values. Up to 75% of the cuticle mass was solubilized by sodium dodecyl sulfate‐β‐mercaptoethanol. Three similar migrating bands were seen in female and male cuticle extracts on sodium dodecyl sulfate–polyacrylamide gel electrophoresis; however, none of the three weaker bands from sporophyte cuticles comigrated with those from gametophytes. Chloroform‐methanol extraction removed < 3.3% of the cuticle mass, suggesting that lipids were minor components.