Formation of the arthrodial membrane in the blue crab,Callinectes sapidus

In this study the pattern of arthrodial membrane deposition in Callinectes sapidus was determined by histological and ultrastructural examination of tissues from the carpus joint of the cheliped collected during premolt, ecdysis, postmolt, and intermolt. Apolysis in the arthrodial membrane occurs at stage D(0) and is synchronous with apolysis of the calcified cuticle. Epicuticle formation begins at early stage D(1) and is completed in late stage D(1). Procuticle deposition starts at D(2) and continues until ecdysis. Numerous cytoplasmic extensions occur throughout the lamellae. Component fibers of the arthrodial membrane are intimately associated with dense plaques on the apical membrane of the underlying hypodermal cells, suggesting a site for fiber polymerization. Deposition of the arthrodial membrane continues after ecdysis, with most of the cuticle thickening occurring during stage C. When stained with PAS and counterstained with hematoxylin, a difference can be discerned between preecdysial and postecdysial procuticle of the arthrodial membrane, a distinction not made in previous studies. The boundary between the arthrodial membrane and calcified cuticle is thicker than either of the two layers and the layers overlap rather than butting up against one another. This pattern suggests that underlying hypodermal cells have to produce multiple types of cuticle over the molt cycle. A summary of the various molting patterns in C. sapidus suggests that the control of these diverse events may prove to be complex.     

Organic and inorganic compound variations in haemolymph, epidermal tissue and cuticle over the molt cycle in Scylla serrata (Decapoda)

Analysis of organic and inorganic compounds in haemolymph, epidermal tissue and cuticle, allowed the correlation of their content variation during the molt cycle in Scylla serrata (Decapoda). The extrusion of carbohydrate, chitin, H(2)O- and NaOH-soluble proteins of the epidermal tissue from D2 stage on suggested an early formation of the organic fraction in the new cuticle. The additional secretion of HCl-soluble protein, glycosaminoglycans, calcium, phosphorus and sulfur in D2-D3 stages suggests mineral nucleation shortly before or at ecdysis. This is consistent with the dominant content of proteins and chitin associated with peaks of carbohydrates and GAG-sulfur groups detected in the cuticle immediately after ecdysis. Furthermore, the maximal phosphorus content at this moment suggests calcium, magnesium and manganese phosphate deposits. After an intense accumulation of calcium, chitin and sharp decrease of protein from A to B stages, a constant rate of their deposition in intermolt and dissolution in premolt occurred. The variations concerning proteins, carbohydrates, glycosaminoglycans, calcium, magnesium and sulfur in the haemolymph, before and after ecdysis, suggest a transitory phenomenon for calcium binding and releasing. Other major elements such as, sodium, potassium and chloride may function as osmotic regulators in the haemolymph and in epidermal compartments. The copper profile presented an indicator role for variations of osmolality over the molt cycle.     

Morphogenesis during molting of the setae in the statocyst sensilla of the mysid shrimp Neomysis integer (Leach, 1814) (crustacea,mysidacea)

The morphogenesis of the setae in the statocyst sensilla of Neomysis integer was studied. Immediately before ecdysis, a new seta lies inverted between the enveloping cells. All of the nine enveloping cells, except the first one, secrete a well defined part of the new seta. The second, third, fourth, and fifth have a trichogen function; the sixth has a trichogen-tormogen function; and the seventh, eighth, and ninth enveloping cells have a tormogen function. It could not be established whether the dendritic sheath is replaced at molt. In the second and third enveloping cells, there is a differential secretion of cuticular material forming the wall of the distal part of the seta. As a consequence, this wall is not homogeneous. The possible role of this heterogeneity in the formation of the gutter-like apical part of the seta is discussed. A mechanism is proposed by which the pore develops at the transition between the midpart and the apical gutter-like part. Before ecdysis, the distal segments of the sensory cells are still connected with the wall of the old seta in the same way as during intermolt. No degeneration is apparent in the distal segments during preparation for the molt. These morphological findings suggest that sensitivity of the sensilla must be maintained until the moment of the ecdysis.     

Studies of the Phoront of Hyalophysa chattoni (Ciliophora,Apostomatida) Encysted on Grass Shrimp1

ABSTRACT. The apostomatous ciliate Hyalophysa chattoni, an ectosymbiont of the grass shrimp Palaemonetes pugio, encysts and dedifferentiates within 48 h from the migratory tomite to a phoretic stage devoid of complex ciliary fields. The presettlement crawling and pivoting of the tomite may play a role in its initial attachment to the shrimp. Metamorphosis of exuviotrophic apostomes has been previously observed to take place immediately prior to host ecdysis. The study has found that Hyalophysa's metamorphosis to the feeding stage on grass shrimp is initiated by a cue from the premolt host and begins during earlier stages of the molt cycle (D₀ and D₁). Due to the long premolt stage of the host's diecdysic molt cycle, metamorphosis is initiated well before ecdysis (over six days). Hyalophysa was able to encyst and metamorphose within 41/4 h when exposed to shrimp in a late premolt stage, indicating that the control of apostome metamorphosis is solely host-dependent.     

Mineral Deposition in Bacteria-Filled and Bacteria-Free Calcium Bodies in the Crustacean Hyloniscus riparius (Isopoda: Oniscidea)

Crustacean calcium bodies are epithelial sacs which contain a mineralized matrix. The objectives of this study were to describe the microscopic anatomy of calcium bodies in the terrestrial isopod Hyloniscus riparius and to establish whether they undergo molt-related structural changes. We performed 3D reconstruction of the calcium bodies from paraffin sections and analyzed their structure with light and electron microscopy. In addition, we analyzed the chemical composition of their mineralized matrices with micro-Raman spectroscopy. Two pairs of these organs are present in H. riparius. One pair is filled with bacteria while the other pair is not. In non-molting animals, the bacteria-filled calcium bodies contain apatite crystals and the bacteria-free calcium bodies enclose CaCO₃-containing concretions with little organic matrix. During preparation for molt, an additional matrix layer is deposited in both pairs of calcium bodies. In the bacteria-filled calcium bodies it contains a mixture of calcium carbonate and calcium phosphate, whereas only calcium carbonate is present in bacteria-free calcium bodies. After ecdysis, all mineral components in bacteria-free calcium bodies and the additional matrix layer in bacteria-filled calcium bodies are completely resorbed. During calcium resorption, the apical surface of the calcium body epithelium is deeply folded and electron dense granules are present in spaces between epithelial cells. Our results indicate that the presence of bacteria might be linked to calcium phosphate mineralization. Calcium bodies likely provide a source of calcium and potentially phosphate for the mineralization of the new cuticle after molt. Unlike other terrestrial isopods, H. riparius does not form sternal CaCO₃ deposits and the bacteria-free calcium bodies might functionally replace them in this species.     

Cuticle Formation in Hemicycliophora arenaria,Aphelenchus avenae and HirschmannIella gracilis

The onset of molting in all stages of Hemicycliophora arenaria was preceded by the appearance of numerous, discrete globular structures which were termed "molting bodies" because they were present in the hypodermis only during the production of the new cuticle. In all parasitic stages the molt commenced with the separation of the cuticle from the hypodermis from which the new sheath and cuticle were differentiated. Following completion of the new sheath and cuticle most of the old outer covering was apparently absorbed before ecdysis. Electronmicrographs of body wall cross sections in molting L4 male specimens revealed the final molt to be a double molt in which an additional sixth cuticle was produced. Since both a new sheath and cuticle were produced during the molt of each stage, the sheath must be considered as an integral part of the cuticle and not as a residual cuticle or the result of an incomplete additional molt. Molting in Aphelenchus avenae and Hirschmanniella gracilis was less complex and "molting bodies" were not observed. After cuticle separation the hypodermis gave rise to a new trilaminate zone, the future cortex, and (later) the matrix and striated basal layers.     

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Molting hormone content,cuticle growth and gastrolith growth in the molt cycle of the crayfishOrconectes limosus

Summary The molting hormone titer during the course of the molt cycle of the crayfish has been determined using an improved extraction procedure. A slight increase of titer is apparent with the transition from stage C (0.29 ng/g) to D_o (0.84 ng/g) when formation of the gastroliths starts. A further increase up to 1.9 ng/g occurs in D₁. A steep, significant increase in hormone content was observed in transition to D₂ when the formation of the new exocuticle commences, the value in D₂ being 56.9 ng/g. The hormone content is only slightly lower in D₃ (39.1 ng/g) when the exocuticle continues to be secreted. A sharp decrease is already observable before ecdysis, in D₄ (9.5 ng/g). The values continue to decrease through stages A (0.84 ng/g) and B (0.29 ng/g), with gastroliths being resorbed. Cuticle growth, however, continues. Thus it is apparent that there are two phases of cuticle growth, a preecdysial phase which is hormone dependent and a postecdysial phase which seems to be independent of ecdysones.We are grateful to Mrs. Brigitte Foertsch for technical assistance and express our gratitude to Dr. Hocks, Schering A.G., Berlin, Germany, for providing the -ecdysone used in this study.     

Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta

When fat body mRNA from the tobacco hornworm larva, Manduca sexta, was translated in a rabbit reticulocyte lysate system, three major polypeptides were found, each having a different developmental profile. One mRNA coded for a 74 kilodalton (K) polypeptide doublet precipitated by an antibody to the arylphorin (manducin). This mRNA was present only during the intermolt feeding phase of the penultimate and the final larval instars. Its appearance 16–24 hr after larval ecdysis was dependent upon the incoming nutrient supply and independent of the juvenile hormone (JH) level. Immunoblots of proteins of the fat body, epidermis, and cuticle revealed the presence of arylphorin in all three tissues. Additionally, several small polypeptides that cross-reacted with the arylphorin antibody were found in the fat body during and up to 24 hr after the last larval molt and in the tanning pupal cuticle. The larval epidermis was also found to contain a small amount of arylphorin mRNA. At the time of the JH decline prior to the onset of metamorphosis, a female-specific mRNA coding for a 79 K translation product appeared. In allatectomized larvae this mRNA was detectable earlier, and its appearance in intact larvae was prevented by application of methoprene, indicating that JH regulates its appearance. At wandering a new mRNA that also codes for a 79 K polypeptide appeared in both sexes and was the major messenger present during the prepupal stage. Neither it nor the female-specific mRNA were translatable after pupal ecdysis.     

The essential role of bursicon during <Emphasis Type="Italic">Drosophila</Emphasis>development

Background  

The protective external cuticle of insects does not accommodate growth during development. To compensate for this, the insect life cycle is punctuated by a series of molts. During the molt, a new and larger cuticle is produced underneath the old cuticle. Replacement of the smaller, old cuticle culminates with ecdysis, a stereotyped sequence of shedding behaviors. Following each ecdysis, the new cuticle must expand and harden. Studies from a variety of insect species indicate that this cuticle hardening is regulated by the neuropeptide bursicon. However, genetic evidence from Drosophila melanogaster only supports such a role for bursicon after the final ecdysis, when the adult fly emerges. The research presented here investigates the role that bursicon has at stages of Drosophila development which precede adult ecdysis.     

Linear intermolt growth in Americamysis bahia (Molenock 1969) (Mysida,Mysidae)

Two phases of allometry, prepubertal, and pubertal, are evident in A. bahia, equally dividing the first six instars. Coefficients of variation of the sizes of individuals’ abdomens, carapaces, and standard lengths significantly decrease in intermolt four, suggesting a synchronization of the size at which individuals enter the pubertal phase. Growth in standard length is best expressed over the first six post-larval instars by a quadratic equation, but within instars 85%, or more, of the expansion occurs linearly during the intermolt period, with only about 15% occurring during ecdysis. Estimations of carapace:abdomen allometry indicate an average ratio of 1:1.97 during the intermolt and 1:1.68 during ecdysis. The average molt:intermolt ratio in the carapace expansion is 1:3.16, and in the abdomen, 1:3.71, indicating that proportional growth between the carapace and the abdomen is continuous during both the intermolt and molt phases, but occur at different rates and proportions during the two periods.     

Molting and cuticle deposition in the subterranean trichoniscid Titanethes albus (Crustacea, Isopoda)

Terrestrial isopods are a suitable group for the study of cuticle synthesis and calcium dynamics because they molt frequently and have evolved means to store calcium during molt. Little data is currently available on molting in Synocheta and subterranean isopods. We studied the molting dynamics in the subterranean trichoniscid Titanethes albus under laboratory conditions and performed a microscopic investigation of sternal CaCO(3) deposits and the tergal epithelium during molt in this species. In accordance with its lower metabolic rate, molting in the laboratory is roughly 2-3 times less frequent in Titanethes albus than would be expected for an epigean isopod under similar conditions. Animals assumed characteristic postures following the molt of each body half and did not consume the posterior exuviae after posterior molt. The structure of sternal calcium deposits and the ultrastructural characteristics of the epidermis during cuticle formation in Titanethes albus are similar to those described in representatives of Ligiidae. During the deposition of the exocuticle, the apical plasma membrane of epidermal cells forms finger-like extensions and numerous invaginations. In the ecdysial space of individuals in late premolt we observed cellular extensions surrounded by bundles of tubules.     

Comparative study of the molting cycle of wild and reared swimming crabs Callinectes danae (Crustacea: Portunidae)

Specimens of Callinectes danae Smith 1869 were caught in northeastern Brazil (2178 males, 2031 females); of these, 24 males and 24 females were reared for 6 months. Water temperature (T) and salinity (S) showed a significant effect on the average ecdysis in wild crab (μ), with a model obtained that was: μ = exp(?0.12268T + 0.06148S)/(1 + exp(?0.12268T + 0.06148S)). Size at morphometric maturity was significantly larger for wild males and females (9.45 and 8.38 cm, respectively) than for reared individuals (8.95 and 7.93 cm). Females of sizes above CW₅₀ (carapace width at maturation) showed an increased ecdysis activity, whereas males showed a decrease in ecdysis frequency in sizes over CW₅₀. Five and six molts were observed for females and males, respectively, in both wild and reared crabs; the modal classes of the reared crabs were shifted to smaller sizes. In reared females the terminal‐pubertal molt occurred at 107 days of age and at 148 days, on average, in males. The intermolt period varied from 8 to 41 days and increased with age.     

High Laccase2 expression is likely involved in the formation of specific cuticular structures during soldier differentiation of the termite Reticulitermes speratus

Termite soldiers are morphologically specialized for colony defense. Analysis of the mechanisms of soldier differentiation is important for understanding the establishment of termite societies. Soldiers differentiate from workers through a presoldier stage and have well-sclerotized and pigmented cuticles. These characteristics are important for nest defense and are likely to be caused by soldier-specific mechanisms of cuticular tanning. The molecular mechanisms leading to cuticular tanning have not been elucidated. Laccase2 (Lac2) plays important roles in this process in insects, and we hypothesized that Lac2 expression may be involved in soldier-specific cuticular tanning. We observed inner and outer head cuticle changes and compared the Lac2 expression patterns among three molts (worker–worker, worker–presoldier and presoldier–soldier) in the termite Reticulitermes speratus. Quantitative analyses of head cuticle colors showed that the color properties changed more conspicuously in presoldier–soldier molts than in the other two molts. Histological observations showed that the exocuticles of soldier heads were substantially thicker than those of worker and presoldier heads, underwent tanning before or just after ecdysis, and were pigmented at earlier time points than other molts. Finally, markedly higher Lac2 expression levels were observed just before and after ecdysis only in the presoldier–soldier molt. These results suggest that specific cuticular formation occurs in the exocuticles during soldier differentiation, and that the high level of Lac2 expression during the presoldier–soldier molt is related to soldier-specific cuticular tanning. We speculate that evolution of the regulatory mechanisms of Lac2 expression were important for the acquisition of soldier-specific cuticles.     

STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM, BOMBYX MORI : III. Ultrastructural Changes of Posterior Silk Gland Cells in the Fourth Larval Instar

The development of the cells in the posterior silk gland of the silkworm, Bombyx mori, during the fourth larval instar has been studied. In the early stages of this instar, the wet weight of the gland and the amounts of RNA, DNA, and protein per animal increase logarithmically until they reach a stationary state at about 72 hr. At around 96 hr of the fourth instar, the larvae enter the molting state, which lasts for about 24 hr until the fourth ecdysis. Towards the end of the molt stage, the growth of the silk gland is resumed. Electron microscopical observation shows that in the early intermolt stage the cytoplasm is filled with free ribosomes and with rough endoplasmic reticulum (ER), first of the lamellar type (0–6 hr) and then of the vesicular or tubular type. The Golgi apparatus also is well developed. At the beginning of the molt stage (90–96 hr), however, most of the ER becomes lamellar in type, concentric lamellar structures being occasionally observed, and the Golgi vacuoles disappear. Autophagosomes and lysosomes increase markedly and the apical portion of the cytoplasm becomes extensively vacuolated; this suggests that the secretory activities are completely depressed, and pronounced degenerative changes appear during the molt stage. Towards the end of the molt stage, large lamellar ER elements are fragmented into smaller lamellae and there is a pronounced increase in the number of free ribosomes.     

Organization of a phyllobranchiate gill from the green shore crab Carcinus maenas (Crustacea,Decapoda)

Summary The phyllobranchiate gills of the green shore crab Carcinus maenas have been examined histologically and ultrastructurally. Each gill lamella is bounded by a chitinous cuticle. The apical surface of the branchial epithelium contacts this cuticle, and a basal lamina segregates the epithelium from an intralamellar hemocoel. In animals acclimated to normal sea water, five epithelial cell types can be identified in the lamellae of the posterior gills: chief cells, striated cells, pillar cells, nephrocytes, and glycocytes. Chief cells are the predominant cells in the branchial epithelium. They are squamous or low cuboidal and likely play a role in respiration. Striated cells, which are probably involved in ionoregulation, are also squamous or low cuboidal. Basal folds of the striated cells contain mitochondria and interdigitate with the bodies and processes of adjacent cells. Pillar cells span the hemocoel to link the proximal and distal sides of a lamella. Nephrocytes are large, spherical cells with voluminous vacuoles. They are rimmed by foot processes or pedicels and frequently associate with the pillar cells. Glycocytes are pleomorphic cells packed with glycogen granules and multigranular rosettes. The glycocytes often mingle with the nephrocytes. Inclusion of the nephrocytes and glycocytes as members of the branchial epithelium is justified by their participation in intercellular junctions and their position internal to the epithelial basal lamina.     

Fine structure of the spermatheca and of the accessory glands in Orchesella villosa (Collembola, Hexapoda)

The spermatheca and the accessory glands of the collembolan Orchesella villosa are described for the first time. Both organs exhibit ultrastructural differences, according to the time of the intermolt in which the specimens were observed. A thick cuticular layer lines the epithelial cells of the accessory glands. In the reproductive phase, they are involved in secretory activity; a moderately dense secretion found in the apical cell region opens into the gland lumen. Cells with an extracellular cistern are intermingled with the secretory cells. These cells could be involved in fluid secretion, with the secretory product opening into the cistern which is filled with an electron-transparent material. After the reproductive phase, the gland lumen becomes filled with a dense secretion. The accessory gland secretion may play a protective role towards the eggs. The spermatheca is located between the accessory glands; its epithelium is lined by a thin cuticle forming spine-like projections into the lumen and consists of cells provided with an extracellular cistern. Secretory cells, similar to those seen in the accessory glands, are missing. Cells with a cistern could be involved in the production of a fluid secretion determining sperm unrolling and sperm motility.     

La cuticule proctodéale des insectes

Résumé La formation des divers types de cuticule rectale a été suivie au cours de la mue chezKalotermes flavicollis (Isoptera). Dans l'épithélium rectal, l'épicuticule externe est formée de couches alternativement sombres et claires, comme dans les autres types de cuticule; l'épicuticule interne présente d'abord une fine lamellation de sa région superficielle, lamellation qui disparaît ensuite. Sur les papilles rectales, l'épicuticule externe a d'abord le même aspect, mais ensuite des différences apparaissent dans l'évolution des différentes couches; ces différences sont encore plus accentuées dans les dépressions épicuticulaires, où les couches superficielles de l'épicuticule externe disparaissent. Sur le cadre bordant les papilles, le dépôt de l'épicuticule externe est peu différent de ce qu'on observe sur l'épithelium rectal; l'épicuticule interne, très mince, présente là aussi une lamellation temporaire; le reste de la cuticule acquiert d'emblée la structure homogène observée pendant l'intermue. Dans tous les cas, la sécrétion de la cuticule (y compris la sous-cuticule) est terminée au moment de l'exuviation. Les modifications des cellules sont brièvement décrites; les cellules des papilles rectales subissent une dédifférenciation complète de leur surface apicale, mais, sur les faces latérales, les associations des mitochondries avec les replis des membranes plasmiques subsistent en grande partie.

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Proctodeal cuticle of insectsII. Formation during the molt

Summary The formation of the different kinds of rectal cuticle has been followed during the molt inKalotermes flavicollis (Isoptera).In the rectal epithelium, the outer epicuticle consists of alternating dark and light layers, as previously described in other types of cuticle; the inner epicuticle first exhibits in its superficial part a thin multilayered pattern, which later disappears.In the rectal papillae, the outer epicuticle has primarily the same appearance, but the evolution of the various layers is somewhat different; even more pronounced differences occur in the epicuticular depressions where the more superficial layers of the outer epicuticle disappear.In the border of the rectal papillae, the outer epicuticle is laid down according nearly the same way as in the rectal epithelium; the temporary multilayered pattern of the very thin inner epicuticle also may be observed; the underlying part of the cuticle, as soon as it is formed, shows the homogeneous appearance observed during the intermolt.In all the cases, the secretion of the cuticle (sub-cuticle included) is achieved when the ecdysis occurs.The modifications affecting the cells are briefly described; the apical surface of the rectal papillae cells undergoes a complete dedifferentiation, but, on the lateral faces, the associations of mitochondria with plasma membrane interdigitations for a large part remain unchanged.