Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation. This work was supported by the German Research Foundation (DFG, grant number MO 1714/1-1).     

Ecdysteroid Titres and Cuticle Depositions in Embryos of the Dipteran Calliphora erythrocephala

Summary

Before the shortening of the germ band, embryos of Calliphora erythrocephala secrete an electron dense layer which resembles a thin atypical cuticle. After completion of the dorsal closure, a second, typical, cuticle is deposited. It is characterized by a thick procuticle and by the presence of hooked setae. This cuticle develops into the larval cuticle of the first instar.

We did not find in newly-laid eggs of Calliphora detectable amounts of either free or hydrolysable conjugated ecdysteroids. A marked rise in ecdysteroid concentrations, essentially attributable to free ecdysone and 20- hydroxyecdysone, occurs shortly before the deposition of the typical cuticle. After a transient decrease, the ecdysteroid titre rises again before hatching.

When eggs are mid-ligatured at blastula or early gastrula stages, the posterior embryonic half is able to build up a typical cuticle with hooked setae. This cuticulogenesis is therefore not dependent on the presence of the embryonic ring glands.     

New spicular skeletons in Turbellaria,and the occurrence of spicules in marine meiofauna1

With Florianella, Bertiliella and Acanthiella, three new genera of Turbellaria are described, one from deep mud bottoms off Norway, and two from sandy bottoms off the south-eastern United States. Whereas the new family Bertiliellidae (within Turbellaria-Eukalyptorhynchia) is established for the two former genera, the latter is of uncertain systematic position, combining features of Archoophora and Prolecithophora-Combinata. All three genera are characterized by a spicular skeleton located in the basement membrane. In the Bertiliellidae the spicules are calcareous (aragonite). A survey of other spiculiferous Turbellaria and meiofauna in general suggests that spicular skeletons, instead of being a special adaptation to the interstitial environment, play a role in body support and tissue economy, and are, in some cases (Turbellaria and Mollusca), possibly relics indicating phylogenetic relationship.     

Cuticle lipids on heteromorphic leaves of Populus euphratica Oliv. growing in riparian habitats differing in available soil moisture

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary‐alcohols and fatty acids. The major cutin monomers were 10,16‐diOH C_16:0 acids. Broad‐ovate leaves (associated with adult phase growth) produced 1.3‐ and 1.6‐fold more waxes, and 2.1‐ and 0.9‐fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane‐synthesis‐associated ECERIFERUM1 (CER1), as well as ABC transporter‐ and elongase‐associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult‐phase broad‐ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited‐moisture environments.     

Cuticle formation in the embryo of Drosophila melanogaster

In Drosophila melanogaster embryos cuticle formation occurs between 12 and 16 hours of development at 25°C. The formation of the cuticulin and the protein epicuticular layers is simultaneous in the hypoderm, the tracheoblasts, and the fore- and hindgut cells. The cuticulin forms as a dual lamina, aggregating from granules secreted by the hypodermal cells. This is followed by the formation of a granular protein epicuticle and finally by the secretion of a mixed fibrous and granular endocuticle. All secretory cells are relatively simple in their ultrastructure. The secretory process is a membrane phenomenon, occurring at the tips of hypodermal microvillae on cells at the surface of the embryo and on those hypodermal cells lining the lumen of the fore- and hindgut. It also occurs along the entire surface of the tracheoblast lumen as well as on the outer surface of those cells which form exoskeletal chitinous setae. The process involves a specialization of the plasma membrane with the formation of secretory granules intracellularly beneath the membrane and the extrusion of these granules through the membrane to the outside where final cuticle formation occurs.     

Cuticle ultrastructure of Hesionid polychaetes (Annelida)

Summary The structure of the cuticle in the four species of the family Hesionidae(Microphthalmus cf.listensis, M. cf.similis, Hesionides arenaria, juv.Podarke spec.) investigated basically corresponds to that found in all annelids. It consists of an outer, electron dense layer, epicuticle, basal cuticle with a fibrous layer, and numerous microvilli which penetrate the layers and are covered by a more or less dense glycocalyx. However, a rough collagen grid is not developed, the fibers are much thinner and are arranged in a more irregular manner. This corresponds to structures found in archiannelids and polychaete larvae. We consider them here to be reductions of the typical polychaete cuticle and postulate a correlation to the small body size of the species investigated. The quantitative differences in cuticle dimensions in the various body regions and structures can also be explained on a purely functional basis, especially apparent in the comparison of prostomium and body trunk. The pharynx cuticle shows significant structural differences due to the development of an additional peripherical lamellar layer-known to this extent only in gastrotrichs—as well as differently shaped and unusually long microvilli. This character is discussed as a possible synapomorphy for the family Hesionidae.

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Zusammenfassung Der Aufbau der Kutikula der 4 untersuchten Species aus der Familie Hesionidae(Microphthalmus cf.listensis, M. cf.similis, Hesionides arenaria, juv.Podarke spec.) entspricht grundsätzlich den Verhältnissen bei allen Anneliden: äußere elektronendichte Schicht, Epikutikula, basale Kutikula mit Faserschicht und zahlreiche Mikrovilli, die diese Schichten durchbrechen und von einem mehr oder weniger dichten Glykokalyx bedeckt sind. Ein derbes Kollagengitter ist jedoch nicht ausgebildet; die Fibrillen der Faserschicht sind wesentlich feiner und unregelmäßiger angeordnet. Dies entspricht Strukturen, wie sie bei Archianneliden und bei Polychaetenlarven gefunden werden. Wir deuten sie hier als Reduktionen der typischen Poly chaetenkutikula und vermuten eine Beziehung zur geringen Körpergröße der untersuchten Arten. Rein funktionell lassen sich auch die quantitativen Unterschiede in den verschiedenen Bereichen der Körperoberfläche deuten, die besonders im Vergleich von Prostomium und Rumpf zum Ausdruck kommen. Die Pharynxkutikula zeigt starke strukturelle Abweichungen durch die Ausbildung einer zusätzlichen peripheren Lamellenschicht (in diesem Ausmaß nur von den Gastrotrichen bekannt) und abweichend geformter, besonders langer Mikrovilli. Dieses Merkmal wird als mögliche Synapomorphie für die Familie Hesionidae diskutiert.

     

Structure of the Cuticle of Metadasynemoides cristatus (Chromadorida: Ceramonematidae)

Structure of the cuticle of Metadasynemoides cristatus (Chromadorida: Ceramonematidae) is examined by light, scanning, and transmission electron microscopy. The nematode has more than 600 annuli, and each annulus has eight cuticular plates. Eight longitudinal ridges, beginning on the cephalic capsule, extend the whole length of the body. Where a ridge traverses an annulus, it forms a complicated articulating structure of overlapping vanes. Within the electron-dense cortical layer, from which the cuticular plates are formed, there are spaces crossed by fine fibrillae, forming what have been termed "vacuoles" by light microscopists. There is an epicuticle and a continuous lucent basal layer. There appears to be no median layer. The cuticle lining of the esophagus and that forming the circum-oral ridge is of much simpler construction.     

Cuticle micromorphology of Saxegothaea (Podocarpaceae)

The cuticle micromorphology of the leaves of the monospecific genus Saxegothaea (Podocarpaceae) was studied by scanning electron microscopy. The external and internal features of the adaxial and abaxial surfaces were characterized. The leaves are hypostomatic. The external adaxial cuticle is rugose with irregular ridges and shallow trench‐like structures that do not correspond to any feature of the inner cuticle surface. The external abaxial cuticle has densely crowded stomata arranged in two bands. The stomata are sunken with pronounced, interrupted Florin rings. Stomatal plugs were not observed. Internally, the adaxial epidermal cells are usually rectangular to square; the abaxial epidermal cells are mainly restricted to the midrib and margins and narrowly rectangular; any among the stomata are irregularly shaped. The stomata are nearly all in direct contact. They show unusual features, including an extra pair of cuticular flanges between the guard cell flanges and those of the lateral subsidiary cells, and ‘bridges’ of lateral subsidiary cell tissue extending polewards above the polar extensions to unite with those at their tips. Neither of these features has been reported previously in Podocarpaceae. The results are discussed in the light of recent phylogenetic studies. It is concluded that, despite its unique cuticular features, Saxegothaea should continue to be regarded as a member of Podocarpaceae. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 58–67.     

Fine structure and metamorphosis of the wax gland cells in a psyllid insect,Anomoneura mori schwartz (Homoptera)

The fine structure of the wax gland of Anomoneura nymph and its metamorphic change were investigated. In the nymph, this organ encircles the anus, and consists of two kinds of cells, derived from epidermal cells: (1) very tall, slim wax cells, which produce and secrete the wax, and (2) flat interstitial cells found among the wax cells. The whole gland is covered by a wax-secreting cuticle with a delicate surface sculpture. Each wax cell has a long, wide duct which opens at the cuticle and penetrates the entire cell. Its cytoplasm is rich in mitochondria and smooth endoplasmic reticulum while that of interstitial cells contains rough endoplasmic reticulum. During each nymphal molt, the cluster of primordial wax gland cells — derived from the epidermis — proliferates rapidly and forms the gland of the next instar. The gland of the preceding instar meanwhile degenerates. Interstitial cells play an important role in cuticle formation and shedding at each molt. These cells alone produce and deposit the new cuticle of the next instar; the wax cells, specialized for wax production, cannot produce cuticle. The apical portion of the wax cell is cut off from the main cell body by growth of the surrounding interstitial cells. Thereafter, the wax cells degenerate, resulting in the rapid disappearance of the previous instar's wax gland. Adults lack this gland entirely.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Cuticle chirality and body handedness in Caenorhabditis elegans

Caenorhabditis elegans adult animals exhibit an inherent chirality of fiber orientation in the basal layer of the cuticle, as well as a naturally invariant but experimentally reversible handedness in the left-right (L-R) asymmetry of the body plan. We have examined the relationship between cuticle chirality and body handedness in normal and L-R reversed animals, using Roller (Rol) mutants and transmission electron microscopy to monitor cuticle properties. Rol phenotypes, several of which have been shown to result from mutations in cuticle collagen genes, are characterized by an invariant, allele-specific handedness in their direction of rolling. We show for several alleles that this direction is not affected by L-R reversal of the body plan. We further show, by electron microscopy, that the chiral orientation of cuticle fibers in animals with normal cuticle is not reversed by L-R body-plan reversal. We conclude that cuticle chirality must be established independently of body-plan handedness. The cues that establish cuticle chirality are still unknown, as are the causes for different rolling directions in different Roller mutants. We discuss the question of how cuticle chirality maintains its independence, and how the orientations of the fiber layers may be determined. Dev. Genet. 23:164–174, 1998. © 1998 Wiley-Liss, Inc.     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Fine Structure of Body Wall Cuticle of Females of Eight Genera of Heteroderidae

Body wall cuticle of adult females of eight genera within the Heteroderidae was examined by transmission electron microscopy for comparison with previously studied species within the family. Cuticle structure was used to test some current hypotheses of phylogeny of Heteroderidae and to evaluate intrageneric variability in cuticle layering. Verutus, Rhizonema, and Meloidodera possess striated cuticle surfaces and have the simplest layering, suggesting that striations have not necessarily arisen repeatedly in Heteroderidae through convergent or parallel evolution. Atalodera and Thecavermiculatus possess similar cuticles with derived characteristics, strengthening the hypothesis that the two genera are sister groups. Similarly, the cuticle of Cactodera resembles the specialized cuticle of Globodera and Punctodera in having a basal layer (D) and a surface layer infused with electron-dense substance. Heterodera betulae has a unique cuticle in which the thickest layer (C) is infiltrated with an electron-dense matrix. Little intrageneric difference was found between cuticles of two species of Meloidodera or between two species of Atalodera. However, Atalodera ucri has a basal layer (E) not found in other Heteroderidae. The most striking intrageneric variation in cuticle structure was observed between the thin three-layered cuticle of Sarisodera africana and the much thicker four-layered cuticle of Sarisodera hydrophila; results do not support monophyly of Sarisodera.     

Cuticle structure and habitat in the Nanorchestidae (Acari: Prostigllnata)

Mites of the cosmopolitan family Nanorchestidae may numerically dominate soil faunas of both hot and cold deserts. The genera Nanorchestes and Speleorchestes differ in their distributions with the former more abundant in cold and the latter in hot regions. We suggest that this is related to differences in cuticular structure in the two genera. The cuticle of Nanorchestes spp. is elaborated with regularly spaced granulations which are absent from Speleorchestes spp. These granulations retain a layer of air over the body which may facilitate cuticular respiration in polar soils that are seasonally waterlogged and increase chances of survival by reducing freezing through direct contact with ice. We discuss the biology of the two genera in terms of the ecological strategies that are selected in hot and cold deserts.     

Some Physical-kinetic Considerations in Penetration of Naphthaleneacetic Acid through Isolated Pear Leaf Cuticle

The penetration of naphthaleneacetic acid (NAA) through enzymatically isolated pear leaf cuticle (Pyrus communis L. cv. Bartlett) is reported herein. Penetration of NAA increased with increasing lime and attained a steady state in approximately 20 minutes. The quantity of NAA penetrating was directly related to the concentration of the donor solution. NAA that penetrated the cuticle was shown to he unaltered. The Penetration of NAA from inside to outside is similar to that from outside to inside. Isolated stomatous lower cuticle permitted approximately 10-foId greater penetration of NAA than the astomatous upper cuticle. The penetration of NAA through isolated pear leaf cuticle is highly temperature dependent, exhibiting a temperature coefficient (Q₁₀) of about 5.6 between 15° and 25 C. The low quantities of chemicals penetrating through the isolated cuticle reported herein and elsewhere are considered to he a characteristic of the technique and not an absolute limitation of the cuticle. Cuticular penetration could account for physiological quantities of NAA entering the plant.     

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus–cuticle interactions related to substrate utilization and resistance to host‐specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell‐free filtrate, for which activity of elastase, N‐acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N‐glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.     

Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

The cuticle is a biological composite material consisting principally of N‐acetylglucosamine polymer embedded in cuticular proteins (CPs). CPs have been studied and characterized by mass spectrometry in several cuticular structures and in many arthropods. Such analyses were carried out by protein extraction using SDS followed by electrophoresis, allowing detection and identification of numerous CPs. To build a repertoire of cuticular structures from Bombyx mori, Apis mellifera and Anopheles gambiae the use of SDS and electrophoresis was avoided. Using the combination of hexafluoroisopropanol and of a surfactant compatible with MS, a high number of CPs was identified in An. gambiae wings, legs and antennae, and in the thoracic integument cuticle of Ap. mellifera pupae. The exoskeleton analysis of B. mori larvae allowed to identify 85 CPs from a single larva. Finally, the novel proteomics approach was tested on cuticles left behind after the molt from the fourth instar of Acyrthosiphon pisum. Analysis of these cast cuticles allowed to identify 100 Ac. pisum CPs as authentic cuticle constituents. These correspond to 68% of the total putative CPs previously annotated for this pea aphid. While this paper analyzes only the recovered cuticular proteins, peptides from many other proteins were also detected.     

Disappearance of the Cuticle and Wax in Outermost Layer of Callus Cultures and Decrease of Protective Ability against Microorganisms

In electron microscopic observation, neither wax nor cuticle was observed on the outermost layers of callus tissues. Chemical estimation of wax in the callus surface was attempted by thin-layer chromatography of solvent extracts of callus tissues in comparison with those of barley and rice leaves. Hydrocarbons and free alcohols were detected in lyophilized callus tissues, but no wax esters or ketones were detected. Germination test indicated that germination of spores of Aspergillus oryzae was less favored on hydrophobic membranes than that of spores of Alternaria sp. and Botrytis cinerea.

From these results, we inferred that the lack of cuticle and wax in the outermost layer of callus tissues facilitated spore germination and penetration, and A. oryzae, a saprophytic fungus, could also readily penetrate into callus tissues.     

Electron Microscopy of the Epidermis and Cuticle of the Needles of Pinus nigra var. maritima in relation to infection by Lophodermella sulcigena

Changes in the ultrastructure of the epidermis and cuticle ofCorsican pine (Pinus nigra var. maritima (Aiton) Melville) inrelation to infection of the needle by Lophodermella sulcigena(Rostr.) v Hohn were recorded throughout a growing season. Epidermalcells when young had thin walls and a thin smooth cuticle: bothstructures thickened with age, and the needle surface becamerough. Densely packed wax tubes developed around the stomatalpore and later became scattered over the needle surface. Themicro-flora present on the needles is briefly noted. Changesin the structure and micro-flora of needles as they aged arethought to contribute to the confinement of infection by L.sulcigena to the needle bases.