The fine structure and deposition of the larval cuticle of the sheep blowfly (Lucilia Cuprina)

The cuticle of Lucilia is composed of an untanned endocuticle and a complex epicuticle of four layers, superficial layer, outer epicuticle, cuticulin and dense layer. The outer epicuticle and attached epicuticular filaments are resistant to acid hydrolysis. During deposition of the cuticle of each larval instar, the cuticulin and dense layers are formed first, followed by the outer epicuticle, which appears to be laid down by secretions from the epidermis passing through the cuticulin via epicuticular filaments. The outer epicuticle is found in the position normally occupied by the wax layer of other insect species.     

Die Feinstruktur des Integumentes und der Muskelansatzstellen vonEchiniscoides sigismundi (Heterotardigrada)

The structure of the integument and the muscle attachments of the marine heterotardigradeE. sigismundi (M. Schultze) was studied by electron microscopy. The cuticle consists of several layers: an outer tripartite (or multilayered) epicuticle, perhaps with an outermost coat; a homogeneous inner epicuticle; a trilaminated layer; an intracuticle; and a fibrous procuticle. These features resemble the cuticle described in Eutardigrada; in contrast, areas on the legs and near the claws, with an outer multilayered epicuticle and a striated layer (inner epicuticle), are — as far as investigated — more similar to the cuticle in Heterotardigrada. The epidermis consists of a single cell layer without glands. The muscle attachments are in line with the general pattern described in the eutardigradeMacrobiotus hufelandi and in Arthropoda.     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.     

An ultrastructural study of the cuticle in the marine annelid Heterodrilus (Tubificidae, Clitellata)

The ultrastructure of the cuticle in four species of the marine Heterodrilus (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus) is investigated with transmission electron microscopy. The noncellular cuticle consists of several parts; closest to the epidermis is a thick zone of collagen fibers embedded in a matrix. The matrix continues outside the fiber zone, forming a layered epicuticle. The external surface of the epicuticle is covered by evenly distributed, membrane-bound bodies, termed epicuticular projections. The epicuticular projections have their longitudinal axis perpendicular to the surface of the cuticle and are attached to the surface by either the surrounding membrane itself or by short pedestals. Microvilli, extensions from the epidermal cells, penetrate and sometimes pass completely through the cuticle. There is interspecific variation in the morphology of the cuticle. The four studied species differ in the arrangement of the collagen fibers, from irregularly distributed fibril bundles to orthogonally arranged fiber layers, as well as in the number and density of layers in the epicuticle. One of the studied species, H. paucifascis, shows intraspecific variation, which is associated with sample locality. The Bahamian specimens of H. paucifascis have four layers in the epicuticle, club-shaped epicuticular projections, and collagen fibers forming a less defined orthogonal grid, while the Belizean specimens have three layers in the epicuticle, epicuticular projections with a bulging part at midlevel, and a distinct orthogonal grid. Based on these findings the variation in the morphology of the cuticle appears to be dependent on both phylogenetic constraints, and functional and environmental factors.     

Cuticular ultrastructure in some marine oligochaetes (Tubificidae)

Abstract. The ultrastructure of the thin, non-cellular cuticle is described for 6 marine oligochaetes, representing 3 of the subfamilies (Phallodrilinae, Limnodriloidinae, and Rhyacodrilinae) of the Tubificidae. The main components of the cuticle in these 6 species, as in most other oligochaetes examined, are: (1) a fiber zone closest to the epidermis, consisting of collagen fibers embedded in a matrix, (2) an epicuticle, which is a continuation of the matrix outside the fiber zone, and (3) epicuticular projections, which are membrane-bound bodies covering the outer surface of the epicuticle. The projections are probably formed by the microvilli that penetrate the cuticle from the epidermal cells below, but this was confirmed only in the studied limnodriloidines. Three of the species examined, Duridrilus turdus, Olavius vacuus , and Heterodrilus paucifascis , lack microvilli. The morphology of the components in the cuticle differs between the studied species. The collagen fibers may form an "orthogonal grid" (i.e., layers of parallel fibers perpendicular to the layers immediately above and below), or they may form parallel layers, or be irregularly scattered. The number of dense layers in the epicuticle, as well as the shape and internal structure of the epicuticular projections, also vary. All these characters might be useful in future phylogenetic analyses to achieve better hypotheses of relationships within oligochaetes as well as to other groups.     

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.     

Robust support for tardigrade clades and their ages from three protein-coding nuclear genes

Abstract. Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).     

First evidence of achiasmatic male meiosis in the water bears Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

Chromosome behaviour during male meioses has been studied in two bisexual amphimictic populations of two tardigrade species, namely Richtersius coronifer and Macrobiotus richtersi (Eutardigrada, Macrobiotidae). Both bisexual populations exhibit a diploid chromosome number 2n=12 and no sex chromosomes were identified. DAPI staining and C-banding data indicate that all chromosomes of the bisexual population of R. coronifer are acrocentric. In both species, at male meiotic prophase, all six bivalent homologous chromosomes are aligned side by side along their length and show no evidence of chiasmata. However, in the oocytes of both species a chiasma is generally present in each bivalent at diplotene stage. Lack of recombination is previously unknown in tardigrades, but is a well known phenomenon in many other metazoans where it is always restricted to the heterogametic sex. In tardigrades there is no evidence of heterochromosomes, but it does not mean that in tardigrades, the heterogametic sex does not exist. The adaptive and evolutionary significance of achiasmatic meiosis is discussed.     

Ultrastructural localization of tyrosinase in the tardigrade cuticle

Tyrosinase activity has been demonstrated ultra-structurally in the cuticle of the eutardigrade Macrobiotus hufelandi. The enzyme could be localized in the outer layer (=epicuticle) of the integument. A weak deposition of reaction product was also seen in the cytoplasm of storage cells, free floating in the haemolymph.     

Ultrastructure of the penetration of the crayfish integument by the fungal parasite, Aphanomyces astaci, Oomycetes

In the crayfish, Astacus astacus, susceptible to the crayfish plague fungus, penetration of the cuticle by the parasite occurred in the soft cuticle. The zoospore lysed the surface lipid layer, tore it away, and formed an infection peg (germ tube) that penetrated through the epicuticle. A septum was formed in the infection peg, and a hypha was formed below the inner epicuticular surface. In the endocuticle, hyphae grew preferrentially parallel to the surface, occassionally perpendicular to it. Growth direction in relation to cuticle architecture is discussed. Subsequently, some hyphae started to penetrate out through the epicuticle. This process was preceded by the swelling of the hyphal tip touching the inner side of the epicuticle. The hypha penetrating out through the epicuticle was much thicker than the infection peg. Histolytic activity, combined with mechanical penetration, seems to be evident in all stages and levels except in the outward penetration of the epicuticular lipid surface layer, where only mechanical rupture could be seen. Differences in the protoplasmic ultrastructure were found between the spore and the penetrant hyphae. Penetration of the cuticle of a resistant crayfish was essentially identical to that in susceptible ones. However, inward penetration of intact epicuticle was too scarce to allow for ultrastructural studies.     

Structure of the cuticle of the alpine weta,Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae)

Sclerotized cuticle segments from the thorax, dorsal abdomen, and ventral abdomen of the alpine, weta Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae) were examined by light microscopy and by scanning and transmission electron microscopy. An epicuticle, exocuticle (outer and inner), mesocuticle, endocuticle, and deposition layer are present in transverse sections. The epicuticle is further composed of a cuticulin layer and inner epicuticle, the latter being finely laminated and containing narrow wax canals that terminate below the cuticle surface. Openings to dermal gland ducts are visible on the surface as are large setae and smaller sensory pegs. Frozen fractured cuticle reveals the presence of horizontal ducts or channels that run laterally within the cuticle. The structure of weta cuticle is compared with that of the common house cricket and arthropods in general.     

Design of the labial cuticle in Cenocorixa bifida Hung. (Hemiptera: Corixidae) with reference to ionic transport

The surface topography and ultrastructure of the labial cuticle of Cenocorixa bifida were examined by scanning and transmission electron microscopy. The dorsal wall of the labium consists of seven sclerotized transverse bars each displaying two rows of semicircular grooves and pores. The cuticle is about 20 microm thick and is composed of epicuticle and lamellate exocuticle and endocuticle, the latter separated from the underlying epidermis by subcuticle containing amorphous material. The epicuticle is subdivided into an electron-dense very thin outer epicuticle and a homogenous thick inner epicuticle, which is penetrated by grooves. The exocuticle is filled with electron-dense blocks of material, which may provide mechanical support to the labial wall. The elongate epidermal cells display extensive infoldings of the apical plasma membrane (facing the cuticle) and contain abundant mitochondria in the cytoplasm. The presence of deep epicuticular grooves and pores in the thin labial cuticle and extensive apical membrane infolding and abundant mitochondria in the epidermal cells suggest that the labium in C. bifida is the site of osmoregulatory ionic uptake.     

Ultrastructural organization of hypodermis and formation of cuticle in pharate larva of Leptotrombidium orientale (Acariformes: Trombiculidae)

The ultrastructural organization of hypodermis and the process of cuticle deposition is described for the pharate larvae of a trombiculid mite, Leptotrombidium orientale, being under the egg-shell and prelarval covering. The thin single-layered hypodermis consists of flattened epithelial cells containing oval or stretched nuclei and smooth basal plasma membrane. The apical membrane forms short scarce microvilli participating in the cuticle deposition. First of all, upper layers of the epicuticle, such as cuticulin lamella, wax and cement layers, are formed above the microvilli with plasma membrane plaques. Cuticulin layer is seen smooth at the early steps of this process. Very soon, however, epicuticle starts to be curved and forms particular high and tightly packed ridges, whereas the surface of hypodermal cells remains flat. Then a thick layer of the protein epicuticle is deposited due to secretory activity of hypodermal cells. Nearly simultaneously the thick lamellar procuticle starts to form through the deposition of their microfibrils at the tips of microvilli of the apical plasma membrane. Procuticle, as such, remains flat, is situated beneath the epicuticular ridges and contains curved pore canals. Cup-like pores in the epicuticle provide augmentation of the protein epicuticle mass due to secretion of particular substances by cells and to their transportation through the pore canals towards these epicuticular pores. The very beginning of the larval cuticle formation apparently indicates the starting point of the larval stage in ontogenesis, even though it remains for some time enveloped by the prelarval covering or sometimes by the egg-shell. When all the processes of formation are over, hungry larvae with a fully formed cuticle are actively hatched from two splitted halves of prelarval covering.     

Ultrastructure of the wax-gland system in subterranean scale insects (homoptera,coccoidea, margarodidae)

The ultrastructure of wax glands (integumentary, stigmatic, and peristigmatic glands) was investigated in larvae, cysts, and adult females and males of species belonging to the genera Porphyrophora, Sphaeraspis, and Eurhizococcus. The general organization and cytological characteristics are similar for all glands studied. Each gland is composed of a single layer of 8 to 40 cells. The glandular cells are characterized by a very large quantity of smooth endoplasmic reticulum which forms dense zones throughout the cytoplasm, but is always placed near the collecting canals in the presence of mitochondria. Each cell has a central canal reservoir which penetrates it deeply and gives rise to a large number of lateral collecting canals, formed by the invagination of the apical plasma membrane. The canals open into a subcuticular cavity forming a common reservoir in which the secretion is accumulated. This reservoir is covered by a modified cuticle formed from the endocuticle and the epicuticle. The endocuticle is composed of a network of fine tubular structures and has many filaments on its surface. The epicuticle is perforated by numerous pores. There is no cuticular duct. The secretion crosses the cuticle in three successive steps. First, it passes through the filaments, then through fine tubular structures of the endocuticle, and finally through the epicuticular pores.     

Ultrastructural study of the cuticle and epidermis in Pseudochordodes bedriagae

The light-microscopic and ultrastructural characteristics of Nematomorpha (Gordiacea) integument are described. Nine male Pseudochordodes bedriagae specimens were collected in the 1997 spring-summer period from the Sauce Chico stream in the Sierra de la Ventana, province of Buenos Aires, Argentina. Of these samples, two were analyzed using scanning electron microscopy (SEM) and the other two using transmission electron microscopy (TEM) in order to establish their morphological characteristics. The morphology of the three integumentary components (epidermis, cuticle and epicuticle) is described. Comparing our findings with those of previous studies, numerous similarities are pointed out. Some variants found would probably be related more to the various criteria of interpreting cut incidences and/or fractures than to actual differences. We assume that the different zones of the cuticle and epicuticle are segregated at the germinal stratum level by the epidermal cells, which would later undergo a slow process of maturation until their exocytosis at the level of the free epicuticular surface.     

Formation of the hindgut cuticular lining during embryonic development of Porcellio scaber (Crustacea,Isopoda)

The hindgut and foregut in terrestrial isopod crustaceans are ectodermal parts of the digestive system and are lined by cuticle, an apical extracellular matrix secreted by epithelial cells. Morphogenesis of the digestive system was reported in previous studies, but differentiation of the gut cuticle was not followed in detail. This study is focused on ultrastructural analyses of hindgut apical matrices and cuticle in selected intramarsupial developmental stages of the terrestrial isopod Porcellio scaber in comparison to adult animals to obtain data on the hindgut cuticular lining differentiation. Our results show that in late embryos of stages 16 and 18 the apical matrix in the hindgut consists of loose material overlaid by a thin intensely ruffled electron dense lamina facing the lumen. The ultrastructural resemblance to the embryonic epidermal matrices described in several arthropods suggests a common principle in chitinous matrix differentiation. The hindgut matrix in the prehatching embryo of stage 19 shows characteristics of the hindgut cuticle, specifically alignment to the apical epithelial surface and a prominent electron dense layer of epicuticle. In the preceding embryonic stage – stage 18 – an electron dense lamina, closely apposed to the apical cell membrane, is evident and is considered as the first epicuticle formation. In marsupial mancae the advanced features of the hindgut cuticle and epithelium are evident: a more prominent epicuticular layer, formation of cuticular spines and an extensive apical labyrinth. In comparison to the hindgut cuticle of adults, the hindgut cuticle of marsupial manca and in particular the electron dense epicuticular layer are much thinner and the difference between cuticle architecture in the anterior chamber and in the papillate region is not yet distinguishable. Differences from the hindgut cuticle in adults imply not fully developed structure and function of the hindgut cuticle in marsupial manca, possibly related also to different environments, as mancae develop in marsupial fluid. Bacteria, evenly distributed within the homogenous electron dense material in the hindgut lumen, were observed only in one specimen of early marsupial manca. The morphological features of gut cuticle renewal are evident in the late marsupial mancae, and are similar to those observed in the exoskeleton.     

吉林和湖北缓步动物二新纪录

记述了吉林省和湖北省2个缓步动物新纪录。它们是Macrobiotus harmsworthi Murray,1907;Macrobiotus hufelandi Schultze,1833。2个种同属于缓步动物门(TaNigrada)、真缓步纲(Eutardigrada)、并爪H(Parachela)、大生熊虫科(Macrobiotidae)、大生熊虫属(Macrobiotus)。     

The distribution of phenoloxidases and polyphenols during cuticle formation

The distribution of phenoloxidase and polyphenols have been studied during cuticle formation at the 4th to 5th molt in Colpodes ethius. Cuticular phenoloxidases arise in the epidermis in cisternae of the rough endoplasmic reticulum, pass through the Golgi complex and are transported to the apical face in secretory vesicles. From the cuticular environment some enzyme is pinocytosed and broken down in the apical multivesicular bodies. Phenoloxidase and polyphenols are present during the formation of the cuticulin layer which also reacts as if it were at least partly composed of a phenoloxidase. The rest of the epicuticle incorporates phenoloxidase as it is deposited, particularly that over the dorsal tubercles which later melanize. Polyphenols do not appear until shortly before ecdysis. They are associated with the epicuticular filaments in both epicuticle and presumptive epocuticle. It is proposed that the epicuticular filaments may arise as liquid crystals with a protein component which becomes stabilized like the rest of the cuticle. These structures could provide a channel for the movement of both lipids and quinones to the surface. Phenoloxidases may pass through fibrous cuticle to be deposited as part of the epicuticle but are incorporated in fibrous cuticle scheduled for sclerotization. The time of stabilization is determined by the availability of polyphenols.     

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.     

The Structure and Mineralization of the Carapace of the Crab (Cancer pagurus L.) 3. The epicuticle

Decalcified and undecalcified preparations of the crab Cancer pagurus in the intermoult condition were studied to examine the mineralization and structure of the epicuticle, using light microscopic, electron microscopic, and microradiographic methods. The epicuticle was found to be composed of two layers, one superficial membrane, and one thicker layer, measuring 1-2 μm. From the base layer spines or microtrichia projected. These were approximately 18 μm long and built like the remainder of the epicuticle. The spines and the base layer of the epicuticle contained vertical canals which in undecalcified sections accomodated columns of crystals. These canals were the only location in which minerals occurred in the epicuticle. In decalcified preparations filamentous strands were observed in the canals. Elsewhere in the epicuticular tissue no fibrillar structure was observed. The canals and their contents seemed to extend across the junctional zone between the epicuticle and the exocuticle.