Moult cycle and morphogenesis inHyas araneus larvae (decapoda,majidae), reared in the laboratory

Moult cycle and morphogenesis in larval instars (zoea I, zoea II, megalopa) of the spider crabHyas araneus (L.) were studied in the laboratory. Changes in the epidermis and cuticle were documented photographically at daily intervals to characterize the stages of the moult cycle. Stage A (early postmoult) is a very short period during which the larva takes up water. During late postmoult (B) and intermoult (C) the endocuticle is secreted, and there is conspicuous epidermal tissue condensation and growth. The onset of early premoult (D₀) is characterized by epidermal apolysis, occurring first at the bases of the setae in the telson of zoeal instars or in the rostrum of the megalopa, respectively. Intermediate premoult (D₁) is the main period of morphogenesis, in particular of setogenesis: in the setae of the zoeal telson and carapace there is invagination or (in the zoea II) degeneration of epidermal tissues. Formation of new setae in the interior of epidermal tubules was observed in zoeal maxillipeds and in the antennae of the zoea II and megalopa instars. During late premoult (Stages D_2–4) part of the new cuticle is secreted, and the results of morphogenesis become clearly visible. For technical reasons (rigid exoskeleton) only a preliminary account of the moult cycle in the megalopa can be given. A time schedule is suggested for the stages of the moult cycle. It is estimated that postmoult (A–B) takes ca 9 to 15 % of total instar duration, intermoult (C) ca 22 to 37 %, and premoult (D) ca 48 to 69 %. There is an increasing trend of relative portions of time (% of total instar duration) from instar to instar in Stages A–C (mainly in the latter) and a decreasing trend in Stage D (mainly in D₀ and D_2–4).     

Ultrastructural shape and three-dimensional organization of the intracuticular canal systems in the mineralized cuticle of the green crab Carcinus maenas

Two main self-contained canal systems are present in the crab mineralized cuticle. The first, or fibre canal system, is constituted by simple, unbranched vertical canals containing axially running fibres in close association with myoepidermal junctions. The second, or pore canal system, is composed of procuticular pore canals and epicuticular channels that prolong the procuticular canals. In opposition to widespread opinion, pore canals make up a three-dimensional branched system extending from the apical plasma membrane of the epidermis up to the epicuticle. Branching occurs by projections of lateral horizontal from the vertical canals at the lower level of the pigmented layer and by innumerable ramifications of epicuticular canals. In agreement with Neville's model for insects, vertical procuticular pore canals of crustacean mineralized cuticle, and also fibre canals, exhibit a twisted ribbon structure reflecting the helicoidal arrangement of the horizontal chitin-protein microfibrils.     

Sensitivity of an insect mechanoreceptor during moulting

ABSTRACT. The fine structure and the behavioural threshold for vibration sensitivity of the eight thoracic filiform hairs of Barathra brassicae caterpillars were investigated through an intermoult/moult cycle. Associated with each filiform hair is one bipolar sensory cell and three enveloping cells. The outer dendritic segment terminates in an ecdysial canal in the hair base and a tubular body lies at its distal end. Shortly before apolysis the dendrite elongates. By this means the connection between the sensory cell and the old cuticular apparatus is maintained while the epithelium and the old thoracic cuticle are separating. The new cuticular apparatus of the filiform hair is formed in the second half of the larval stage by the three enveloping cells. A second tubular body in the elongated outer dendritic segment is formed at the base of the replacement hair 10 h before next ecdysis, so that the new hair functions as soon as ecdysis is completed, the old cuticular apparatus with the old tubular bodies being torn away with the exuvia during ecdysis. Sensitivity to a 300 Hz tone was tested in the standing wave of a Kundt's tube. Throughout most of the larval instar the threshold was 2.0 ± 0.3 μm particle displacement amplitude until 1–2h before ecdysis when it rose to 6.8 ± 1.3 μm and at 10–30 min before the beginning of ecdysis no reaction to sound could be detected. Once the old cuticle was shed maximum sensitivity returned as soon as the replacement hairs were erect. The sensilla are therefore physiologically functional at all developmental stages except for 30–60 min during actual ecdysis.     

Ultrastructure of the epidermal maxilla II-gland of Scutigera coleoptrata (Chilopoda, Notostigmophora) and the ground pattern of epidermal gland organs in Myriapoda

The epidermal maxilla II-gland of Scutigera coleoptrata was investigated using light and electron microscopy. The glandular epithelium surrounds a spacious integumental cavity at the base of the maxilla II. The gland is formed as a compound gland organ that is composed of thousands of epidermal gland units. Each of them consists of four different cell types: a secretory cell, an accessory or intermediary cell, and a proximal and distal canal cell. The intermediary and the two canal cells form a conducting canal. Only in the most distal part of the intermediary cell is the canal lined by a cuticle. In the area of the two canal cells, the conducting canal is completely covered by a cuticle. The canal passes through the cuticle and opens into the spacious integumental cavity, which serves as a secretion reservoir. The structural organization of the epidermal maxilla II-gland was compared to that of other compound epidermal gland organs in Chilopoda and Diplopoda. All these glandular organs in Myriapoda share the same ground pattern.     

Calcium,phosphorus and adenylate levels and Na(+)-K(+)-ATPase activities of prawn,Macrobrachium nipponense,during the moult cycle

Changes in calcium and phosphorus concentrations, adenylate (AMP, ADP and ATP) levels, and ratios and ATPase activities of Macrobrachium nipponense were investigated during the moult cycle. Ca level in the exoskeleton was lowest in early postmoult (stage A), increasing at stages B and through intermoult (stage C) and peaking in premoult (stage D1 and D2). The P concentrations in the exoskeleton and muscle in late premoult and early postmoult stages were higher than those at other moult stages, and were lowest in the intermoult. Muscle adenylate energy charge (AEC) changed with moult stages, and was in agreement with the change in inorganic P level in the muscle. AEC may be a direct indicator of energy metabolic activity during the moult cycle. ATP/ADP and ATP/AMP ratios in premoult and postmoult stages were higher than that in intermoult stage. Na(+)-K(+)-ATPase activities of gills, muscles and hepatopancreatic of prawns were higher in early postmoult and late premoult animals, whereas they were lower in late postmoult, intermoult and early premoult animals. Gill residual ATPase activity was significantly higher in postmoult animals, while the peak value of hepatopancreatic residual ATPase activity appeared in intermoult stage.     

Ultrastructural organization of the anal organs in the anal capsule of Craterostigmus tasmanianus Pocock, 1902 (Chilopoda, Craterostigmomorpha)

We describe the ultrastructural organization of the anal organs of Craterostigmus tasmanianus, which are located on the ventral side of the bivalvular anal capsule. Each part of the capsule bears four pore fields with several anal pores. The pores lead into a pore canal, which is surrounded by the single-layered epithelium of the anal organs. Each anal organ is composed of four different cell types: transporting cells of the main epithelium, junctional cells, isolated epidermal glands, and the cells forming the pore canal. The transporting cells exhibit infoldings of the outer cell membranes, forming a basal labyrinth and a poorly developed apical complex. The cells are covered by a specialized cuticle with a widened subcuticular layer. Only the cuticle of the main epithelium is covered by a mucous layer, secreted by the epidermal glands. The ultrastructural organization of the anal organ is comparable to the coxal and anal organs of other pleurostigmophoran Chilopoda. It is likely that the coxal and anal organs of the Pleurostigmophora are homologous, due to their identical ultrastructural organization. Differences concerning the location on the trunk of Pleurostigmophora are not sufficient to reject a hypothesis of homology. Anal organs are found not only in Craterostigmomorpha, but also in most adult Geophilomorpha, and in larvae and most adults of Lithobiomorpha. The anal organs of C. tasmanianus are thought to play an important role in the uptake of atmospheric water. J. Morphol.     

Apolysis and the turnover of plasma membrane plaques during cuticle formation in an insect.

The apical plasma membranes of Calpodes epidermal cells have small fattened areas or plaques with an extra density upon their cytoplasmic face. The plaques are typically at the tips of microvilli. The are present during the deposition of fibrous cuticle and the cuticulin layer. Since the plaques are close (less than 15nm) to the sites where these kinds of cuticle first appear, they are presumed to have a role in their synthesis and/or deposition and orientation. When fifth stage larval cuticle deposition ceases prior to pupation, the plaques are lost as the area of the apical plasma membrane is reduced. The plaques pass from the surface into pinocytosis vesicles and multivesicular bodies where they are presumably digested. The loss of plaques occurs as the blood level of moulting hormone reaches a peak at the critical period after which the prothoracic glands are no longer needed for pupation. Apolysis or separation of the epidermis from the old cuticle is the stage when plaques are absent, the old ones have been lost but the new ones have yet to form. After the critical period, the epidermis prepared for pupation with a phase of elevated RNA synthesis at the end of which plaques and microvilli reform in time to secrete the new cuticulin layer and later the fibrous cuticle of the pharate pupa. There is a new generation of plaques for each moult and succeeding intermoult and each generation is involved in two kinds of cuticle deposition before involution and redifferentiation.     

Untersuchungen zur Häutungsphysiologie der dekapoden Krebse am Beispiel der StrandkrabbeCarcinus maenas

Under constant laboratory conditions, juvenile shore crabs moult at fixed intervals which depend upon their body size. During one moult every crab exhibits increases of the same relative amounts, independent of its absolute size. Basing on the predictable duration of the intermoult period, the morphological changes in the structure of the cuticle and the development of limb-buds, the intermoult period could be divided into 21 different stages. After studying the moulting rhythm in constant milieu, the influence of the following exogenous and endogenous factors upon the moulting rhythm and growth of normal and of eye-stalkless individuals was investigated: temperature, photoperiod, loss of pereiopods, feeding, and presence of larger specimens. From these investigations it became evident that the moulting rhythm is regulated by growth. The crabs are able to moult only after achieving a minimum of tissue growth. So long as this minimum growth is not achieved, a moult-inhibiting hormone is secreted and moulting is prevented. If the moult-inhibiting hormone is absent, moulting hormone is secreted and initiates a moult. Under dangerous conditions, the crabs are able to delay the next moult. Under unfavourable conditions they consume less food than normal. Therefore, the amount of tissue growth which is the necessary prerequisite for moulting is delayed, and continued release of moult-inhibiting hormone prevents the moult. Under conditions favourable for moulting, or demanding moult (e. g. after loss of many pereiopods) the crabs accelerate the moult. Temperature influences the moulting rhythm by indirect effects on the metabolic rate. During further investigations, the variation of the following parameters were determined quantitatively: content of moulting hormone in whole crabs; content of aminoacids, protein, glucose, Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in the hemolymph; pH and osmotic pressure in the hemolymph; and Ca⁺⁺ content in skeleton and whole crabs. All parameters mentioned — excepting pH and K⁺ content of the hemolymph — vary characteristically during the intermoult period. The titre of moulting hormone has 4 different maxima. Of all parameters, only the content of animoacids and protein in the hemolymph vary in the same way as the titre of the hormone. From these results the following conclusions are drawn: The moulting hormone not only initiates the moulting process, but controls it at several stages. Only protein metabolism seems to be under direct control of the moulting hormone which stimulates protein-synthesis. Chitin formation, regeneration, apolysis and ecdysis are indirectly controlled by the moulting hormone through protein metabolism. As in most of the other processes mentioned, the calcification of the new cuticle is not under the direct influence of the moulting hormone. The conclusion ofDigby (1966) that calcification in crabs is an electrochemical process, is confirmed.     

Free amino acids in claw muscle and haemolymph from Australian freshwater crayfish at different stages of the moult cycle

Amino acids were measured in claw muscle and haemolymph in the freshwater decapod crustacean, Cherax destructor, at different stages of the moult cycle. The total pool of amino acids in muscles from animals in intermoult (97+/-13 mmol kg(-1) muscle), premoult (80+/-20 mmol kg(-1)) and postmoult (97+/-19 mmol kg(-1)) were not significantly different. Despite the relatively stable total pool of amino acids, there were changes in the concentrations of alanine, glutamine and proline over the moult cycle. Compared to intermoult, claw muscles from animals in premoult had a lower concentration of proline, and animals in postmoult had higher concentrations of alanine and glutamine, but lower concentrations of proline. Concentrations of alanine and glutamine in claw muscle of animals in postmoult were higher and proline concentrations lower than in the same animals during the premoult stage. The concentration of proline in haemolymph was lower in animals in premoult and postmoult compared to intermoult. The total amino acid pool in the claw muscle of Cherax destructor did not change significantly over the moult which is distinctly different to the changes in amino acids reported in the claw muscles of marine decapod crustaceans.     

Some ultrastructural observations on the integument of a pentastomid

The cuticle of the cephalobaenid pentastomid Reighardia sternae is described at various stages of the moult-intermoult cycle. The intermoult cuticle comprises four layers: an outer epicuticle; an underlying dense layer, the protein epicuticle; a fibrillar endocuticle; and a denser subcuticle. The overall similarity between the structure and composition of these layers and those of insects is discussed. However, the orientation of the chitin-protein fibres in the endocuticle does not show the rotating structure characteristic of many arthropod species, but this does appear in the sclerotized hooks. It is suggested that this comparatively loose, poorly oriented endocuticular structure produces a highly extensible cuticle which is precisely adapted to the specialized, endoparasitic habit of this species. Events at ecdysis, particularly the secretion of moulting fluid and the deposition of cuticulin, follow the insect pattern precisely. The phyletic significance of these observations is discussed.     

Imaginal differentiation of the integument of the desert locust, Schistocerca gregaria Forsk. IV. Steps in morphogenesis of the glandular units

In the gregarious males of Schistocerca gregaria Forsk., imaginal moulting (mitosis, apolysis, new cuticle synthesis, ecdysis) is associated with the differentiation of numerous glandular units. These units, involved in a sexual, excitatory pheromone secretion are at that time composed only of a basal glandular cell and an apical duct cell. Each glandular unit originates from an isogenic group of cells of which the four elements (tetrade) are disposed on two levels. At each level a principal cell and an accessory one may be recognized. The lower accessory, or ciliary, cell shows, at the time of apolysis, both a strong cytoplasmic protrusion and a typical ciliary formation. This formation associated with a diplosome goes through the duct cell and ends up in the exuvial space. It makes a inner mould; arranged around it are epicuticular materials characteristic of the duct wall; then it disappears. The strong cytoplasmic protrusion also retracts thus allowing a glandular reservoir to form. A glandular cell may be recognized at an early stage owing to its R.E.R. development. The upper accessory cell strengthens the duct cell and secretes junctional cuticle between the duct and general cuticle. Accessory cells, after the imaginal moult do not degenerate but acquire epithelial cell characteristics. The duct has a dual origin : the receptive part is secreted by the ciliary cell and the vector part by the duct cell. The organization and stages of morphogenesis of the glandular unit are discussed and compared to those of other apterygote or pterygote insects.     

The fine structure of the carapace integument of Daphnia magna straus (Crustacea branchiopoda)

Summary The structure of the two integumental layers comprising the carapace of female D. magna was examined at several points through the molt cycle. The epicuticle and procuticle are simple in organisation; pore canals are absent but intracuticular fibres are present, forming complexes with invaginations of the epidermal plasma membrane similar to such complexes described in the literature for other arthropods. The epidermis consists almost entirely of cuticle-secreting cells. Secretion of the new cuticle begins when 50–67% of the instar has elapsed by which time the epidermal cells have increased in height and their nuclei have become more rounded. However, other presumed secretory phenomena observed viz. the formation of dense core vesicles by Golgi bodies, and the occurrence of these and coated vesicles near the apical plasma membrane are not restricted to any particular period during the molt cycle. This suggests that the mechanisms of cuticle secretion do not undergo marked changes in activity as they do in decapods; presumably this relative continuity is related to the much shorter molt cycle of cladocerans.The technical assistance of G.A. Bance, and the financial support provided by the National Research Council of Canada are gratefully acknowledged     

The moult cycle of the terrestrial isopod Armadillo officinalis Duméril, 1816 (Crustacea: Isopoda: Oniscidea)

The present work focuses on the moult cycle of Armadillo officinalis. For a 100‐day period, 134 animals were observed and routinely examined with the aim of detecting distinctive morphological characters in the several stages and substages of the moult cycle and of disclosing their duration. Statistical tests and Poisson regression models with robust standard errors were used to investigate differences and relationships between moult and the size and gender of the animals. The appearance of the calcium carbonate deposits on the pereon sternites during the premoult stage was documented in detail, and three main substages were identified. The average duration of the premoult and of the biphasic ecdysis was about 12 and 1.5 days, respectively. This observation period, however, did not allow to establish a determined average duration of the intermoult stage, which was extremely variable. This stage lasted for 2 months or more in most of the cases observed, but about 1‐month‐long intermoult stages were also recorded. No statistically significant association was found between the number of moults and gender and size of the animals.     

Modified pore canals in the cuticle of Gammarus (Crustacea : Amphipoda); A study by scanning and transmission electron microscopy

A novel type of pore canal is described from the cuticle of three species of Gammarus. Each canal passes from the epidermis vertically through the endocuticle and exocuticle, and in the most distal layers of the latter is slightly expanded. Before entering the epicuticle the canal narrows, forming a neck the base of which is encircled by an electron-dense collar. Several tubular structures arise from the collar and pass distally into the reticular innermost regions of the epicuticle. Within the neck and just below its opening at the cuticle surface, a rod-like structure is inserted; this protrudes a short distance from the pore. Each pore canal is connected to many necks; the openings of the latter are aligned in rows over the surface, the openings and rows being about 0.15 and 1.0 μm apart, respectively. Changes in the pore and canal contents are visible and their significance is discussed.     

The structure and growth rates of the cuticle of the stick-insect Heteropteryx dilatata

Locust-style and pseudo-orthogonal structures were found in the cuticle of Heteropteryx dilatata, with the tibiae and femora containing both, and the ocular facets showing only helicoidal construction. The same epidermal cells appear to be competent to secrete both locust-style and pseudo-orthogonal cuticle structure. Curved fibres were found to exist and an apparent 'diagonal lattice' construction of the fibres in the thoracic and abdominal tergites and sternites at about 45 degrees to the insect's long axis. The advantages of flight have apparently been sacrificed for increased 'armour-plating' and the wings exhibit the characteristics of flightlessness. The minimum age post final moult of the insect as calculated from the growth layers was 7 weeks, the actual age being unknown. 'Trauma lines' and 'continuous lamellation' were seen and several possible explanations discussed.     

The transfer of lipid in insects from the epidermal cells to the cuticle

The structure of the pore canals and the tubular filaments they contain are described in a series of insects and types of cuticle. In all these cuticles the tubular filaments arise from the plasma membrane of the epidermal cells and they contain argentaffin material, regarded as sclerotin precursors, and lipid-staining material, regarded as wax precursors. These materials are transferred to the inner epicuticle and are exuded over the surface of the outer epicuticle to form the waterproofing layer as described in the preceding paper. They are also transported to those parts of the endocuticle destined to form hard exocuticle. There are no terminations of tubular filaments in the soft cuticle of Manduca larva, in the soft expanding cuticle of Rhodnius, and in the non-sclerotized post-ecdysial endocuticle of Tenebrio. Apis. etc. In the puparium of Calliphora lipid appears to be added by the epidermal cells directly and not by way of tubular filaments. It is confirmed that lipid is a component of sclerotized cuticle.     

Amorphous and crystalline calcium carbonate distribution in the tergite cuticle of moulting Porcellio scaber (Isopoda, Crustacea)

The main mineral components of the isopod cuticle consists of crystalline magnesium calcite and amorphous calcium carbonate. During moulting isopods moult first the posterior and then the anterior half of the body. In terrestrial species calcium carbonate is subject to resorption, storage and recycling in order to retain significant fractions of the mineral during the moulting cycle. We used synchrotron X-ray powder diffraction, elemental analysis and Raman spectroscopy to quantify the ACC/calcite ratio, the mineral phase distribution and the composition within the anterior and posterior tergite cuticle during eight different stages of the moulting cycle of Porcellio scaber. The results show that most of the amorphous calcium carbonate (ACC) is resorbed from the cuticle, whereas calcite remains in the old cuticle and is shed during moulting. During premoult resorption of ACC from the posterior cuticle is accompanied by an increase within the anterior tergites, and mineralization of the new posterior cuticle by resorption of mineral from the anterior cuticle. This suggests that one reason for using ACC in cuticle mineralization is to facilitate resorption and recycling of cuticular calcium carbonate. Furthermore we show that ACC precedes the formation of calcite in distal layers of the tergite cuticle.     

Ultrastructural investigation of the vesicular glands in Scutigera coleoptrata (Chilopoda,Notostigmophora)

In the notostigmophoran centipedes, two pairs of vesicular glands have evolved. These paired glands are situated in the first and second trunk segment and open via cuticular ducts in the upper part of the particular pleura. The vesicular glands of Scutigera coleoptrata were investigated using light and, for the first time, electron microscopical methods. The glands consist of wide sac‐like cavities that often appear vesicular. The epithelia of both glands are identically structured and consist of numerous glandular units. Each of these units consists of four different cells: a single secretory cell, a small intermediary cell, and one proximal and one distal canal cell. The intermediary cell forms a conducting canal and connects the secretory cell with the canal cells. Proximally, the intermediary cell bears microvilli, whereas the distal part is covered with a distinct cuticle. The cuticle is a continuation of the cuticle of the canal cells. This investigation shows that the ultrastructure of glandular units of the vesicular glands is comparable to that of the glandular units of other epidermal glands in Chilopoda and Diplopoda, although the glands look completely different in the light microscope. Thus, it is likely that the vesicular glands and epidermal glands share the same ground pattern. With regard to specific differences in the cuticular lining of the intermediary cells, a common origin of epidermal glands in Myriapoda and Hexapoda is not supported. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

Ultrastructural location of calcium and magnesium during mineralisation of the cuticle of the shore crab,as determined by the K-pyroantimonate method and X-ray microanalysis

We have investigated the distribution of Ca²⁺ and Mg²⁺ in the new cuticle of moulting shore crabs (Carcinus maenas), using the K-pyroantimonate method in combination with X-ray microanalysis in order to identify antimony precipitates. During the premoult period, Ca²⁺ and Mg²⁺ accumulate in well-defined sites of the new pigmented layer. After moulting, mineralisation appears to begin preferntially at these sites. These form a honeycomb-like structure that quickly increases the rigidity of the new cuticle, with a small recruitment of material from extraneous sources. Mineralisation of the principal layer, on the other hand, immediately follows deposition of the organic matrix. Our experiments also provide evidence that the epidermal cell extensions associated with the pore canals are the means by which Ca²⁺ and Mg²⁺ are transferred from the epidermis into the mineralising cuticular layers. The plasma membrane of these cell extensions appears densely lined by particles of antimony precipitate that probably mark the location of the transporting sites. Shortly after moulting, the distribution of mineral deposits is such that the cell extensions cross the mineralised lamellae of the principal layer and constitute preferential access routes to the pigmented layer, where mineralisation is still in progress.