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.     

The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO₃) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na⁺ followed by Mg²⁺, Ca²⁺ and K⁺. The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO₃ precipitation and dissolution.     

Lipid metabolism of the female crab Pachygrapsus marmoratus during the moulting cycle

The moult induces important variations in the concentration and fatty acids composition of lipid classes during the moulting cycle of the female crab Pachygrapsus marmoratus regardless of the maturational degree of ovaries. Sexual maturity is characterized by a rise in lipids. Juveniles contain high levels of polyunsaturated fatty acids except in the ovary, whereas adults are composed mainly of monoethylenic acids. The moulting decrease of saturated fatty acids shows their importance during this crucial period, owing to their utilization as energy sources at the time of ecdysis. The late premoult fall of hepatopancreatic lipids results at once from inanition during the period before the exuviation and transfer of lipids through the hemolymph to the periphery in order to realize numerous changes occurring during the moulting process. The relative stability of lipid composition of ovaries in sexual pause during the moulting cycle agrees with a reduced metabolism of ovarian cells.     

Spatial distribution of calcite and amorphous calcium carbonate in the cuticle of the terrestrial crustaceans Porcellio scaber and Armadillidium vulgare

The crustacean cuticle is an interesting model to study the properties of mineralized bio-composites. The cuticle consists of an organic matrix composed of chitin–protein fibres associated with various amounts of crystalline and amorphous calcium carbonate. It is thought that in isopods the relative amounts of these mineral polymorphs depend on its function and the habitat of the animal. In addition to the composition, the distribution of the various components should affect the properties of the cuticle. However, the spatial distribution of calcium carbonate polymorphs within the crustacean cuticle is unknown. Therefore, we analyzed the mineralized cuticles of the terrestrial isopods Armadillidium vulgare and Porcellio scaber using scanning electron-microscopy, electron probe microanalysis and confocal μ-Raman spectroscopic imaging. We show for the first time that the mineral phases are arranged in distinct layers. Calcite is restricted to the outer layer of the cuticle that corresponds to the exocuticle. Amorphous calcium carbonate is located within the endocuticle that lies below the exocuticle. Within both layers mineral is arranged in rows of granules with diameters of about 20 nm. The results suggest functional implications of mineral distribution that accord to the moulting and escape behaviour of the animals.     

Effects of ingested phytoecdysteroids on the growth and development of two Lepidopterous larvae

The effects of ingested or injected 20-hydroxyecdysone on silkworm larvae (Bombyx mori) including death without moulting, death following completion of promoted moulting, death during promoted moulting (ecdysis inhibition) and inhibition in growth with and without effects on moulting, are dependent upon the concentration of exogenous hormone, the precise developmental stage of the treated larvae, and the duration of exposure to the exogenous ecdysteroid. Comparisons of 20-hydroxyecdysone with other phytoecdysteroids in the silkworm and pink bollworm, Pectinophora gossypiella, show a similar but more potent effect induced by ponasterone A, while cyasterone causes an ‘antiecdysone’ effect.     

Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locusta migratoria L.

The kinetics of secretion of ecdysone by the prothoracic glands of Locusta migratoria were studied during the last larval instar. Three stages of intense production of ecdysone (α-ecdysone) were monitored during this developmental period: they correspond to three peaks of moulting hormone concentration in the blood, which indicates that the main regulation of the moulting hormone titre is achieved through variations in prothoracic gland activity. In the haemolymph the ratio of ecdysone to ecdysterone (20-hydroxy-ecdysone) is in favour of ecdysone during the two first moulting hormone peaks ecdysterone being by far predominant over ecdysone at the time of the third (major) peak; these results support previous studies on the metabolic fate of injected labelled ecdysone during the same developmental period in Locusta migratoria.     

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.     

Unravelling the moulting degradome: new opportunities for chemotherapy?

Replacement of the nematode cuticle with a newly synthesized cuticle (a process known as moulting) occurs four times during larval development. Therefore, the key components of this essential developmental process represent attractive targets for new chemotherapeutic strategies. Recent advances in understanding the molecular genetics of nematode moulting should stimulate and facilitate development of novel drugs that target the essential molecules of the moulting cycle. In particular, we argue that further understanding of the moulting degradome and its key peptidase members offers an important opportunity for the development of novel antinematode agents.     

Studies on an Indian polydesmoid millipede Streptogonopus phipsoni Life cycle and swarming behaviour of the larvae

The adults of Streptogonopus phipsoni (Pocock), an Indian polydesmoid millipede, emerge from the soil at the onset of the monsoon and mate. Eggs are laid in the soil and the adults die. The larvae leave the soil at stadium III and, apart from moulting periods, remain on the surface until the end of the monsoon. The larvae are aggregated in swarms, usually consisting of several hundred individuals. They are active on open ground, including metalled roads, during daylight, but usually spend the night in an inactive state under cover of stones or vegetation. If a swarm is disturbed, the individuals scatter but later reaggregate.
Laboratory experiments show that the larvae are sensitive to light even though they lack eyes, and if given a choice spend more time in the light than the dark. Just before and after moulting however they spend more time in the dark than in the light.
Experiments were carried out on the reactions of the larvae to aquatic suspensions of benzaldeyde, since this substance is related to components of the exudate of the repugnatorial glands; strong concentrations (10^-1) repelled the larvae, whilst weak concentrations (10^-6) attracted them. It is suggested that swarms may be dispersed or reaggregated by the effects of variations in concentration of components of the exudates. The benefits to the larvae which may result from swarming are discussed.     

Seasonal immunocytochemical and ultrastructural changes in the neurohypophysis of the migratory Canada goose

Immunocytochemical localization of arginine vasotocin (AVT) in the neurohypophysis of the migratory Canada goose was studied during six periods (Spring premigratory, Spring postmigratory, breeding, moulting, Fall or Autumn premigratory, and Fall postmigratory) of its annual life cycle. Ultrastructural changes in the pars nervosa, with emphasis on the axonal endings, were also examined during the above periods. In the Spring premigratory period, the staining intensity for AVT was greater in the anterior median eminence than in the pars nervosa where the density of axonal secretory granules was lower than that in the preceding period, in both sexes. During moulting females seemed to be under greater osmotic stress than males as indicated by low AVT staining intensity and secretory granule concentration in the pars nervosa. The occurrence of considerable amounts of lipofuscin bodies in the pars nervosa of both sexes during moulting was indicative of high lysosomal activity in the pituicytes. AVT release is recognized as a significant event in initiating Spring migration.     

Tissue- and Stage-Specific Expression of Sericin Genes in the Middle Silk Gland of Bombyx mori

Sericin gene expression in the middle silk glands during Bombyx mori larval development was analyzed using probes from a genomic DNA clone for 10.5 kb sericin mRNA. The 10.5 kb mRNA, the most abundant in the fifth instar, is not detected in the third feeding, fourth feeding and fourth moulting stages. It becomes detectable at 2 days of the fifth instar, and accumulates rapidly. The second major mRNA in the late fifth instar, a 9.0 kb component having a similar sequence to the 10.5 kb mRNA, becomes detectable only at 6 and 7 days of the instar by use of the repetitious coding sequence probe of the sericin clone. Using the same probe about 20 kb RNAs with a fainter intensity than that of the major mRNAs are detected. They are present extremely faintly in the third and fourth feeding stages, disappear in the fourth moulting stage, and increase in the fifth instar. Two other faint poly(A)⁺ RNA components are detected by a DNA probe containing the 5' end sequence of the sericin clone. One is 4.3 kb, and appears in the third, fourth and fifth feeding stages but not in the fourth moulting stage. The other is 3.0 kb, and it becomes detectable after 1 day of the fifth instar.     

Lipid metabolism of the male crab Pachygrapsus marmoratus during the moulting cycle

The moult induces important variations in the concentrations and fatty acid composition of lipid classes during the moulting cycle of the male crab Pachygrapsus marmoratus. Phospholipids are the predominant lipid class. The high levels in total saturated fatty acids but also in oleic and palmitic acids result of the semi-terrestrial ethology of this grapsidae. The hepatopancreas presents the most notable variations in the lipid concentration during the moulting cycle. Transport of hepatopancreatic lipids through the hemolymph to the periphery occurs during the end of premolt, just before the new tissues constitution.     

Moulting in nematodes: The formation of the adult cuticle during the final moult of Nippostrongylus brasiliensis

The process of moulting and the formation of the new cuticle during the final moult of the nematode Nippostrongylus brasiliensis have been described. After separation of the hypodermis from the old cuticle, the new cuticle is secreted by the hypodermis. The first layers to be formed are the outer trilaminate membrane and the fibre layers. The struts of the cuticle separate out from the fibrillar and granular components of the outer cuticle. There is no reabsorption of the old cuticle.     

Biochemical Studies on the Mineral Components in Sake Yeast

Analysing the mineral components in yeasts (sake yeast and some of other brewer’s yeasts), the author found that no remarkable difference was seen on the composition of the major mineral components. The potassium content of yeast cultured in koji extract is lower than that cultured in malt extract or in the synthetic medium. Potassium concentration of koji extract is lower than that of each of other media, and it was possible to increase the potassium content of the cells to the normal level by the enrichment of the koji extract with potassium chloride. From these facts, it is assumed that the potassium concentration of koji extract is insufficient for the saturation in the cells with potassium.

Phosphorus and magnesium contents in the cells are not so much affected by pH of the medium as potassium.     

Moulting of mechanoreceptive hair sensilla of Lepisma saccharina (Zygentoma) and Machilis spec. (Archaeognatha)

In all insects investigated hitherto the mechanoreceptive hair sensilla possess the same organization plan. There are differences in the moulting process of hemimetabolous insects. While the functions of the three enveloping cells are corresponding, the position of the moulting porus is different. In Gryllus and Periplaneta the moulting porus is situated at the base of the hair, in Machilis 25 mum above the base of the hair. In Lepisma, however, the moulting porus is located at the tip of the hair shaft. Comparison of mechanoand contactchemoreceptive hair sensilla shows that the position of the moulting porus in primitive ectognathous insects represents an argument for the close morphological and phylogenetical relationship between both types of sensilla. Interpreting our results, the original position of the moulting porus is at the tip of the hair sensilla. In mechanoreceptors, where the moulting porus is located at the base of the hair, the original tip of the hair (position of the moulting porus) is overgrown at the side by a 'secondary hair process'.     

The gastrin-secreting effects of the salt components of naftusia water]

Intragastric administration of certain salt components of mineral water naftusia in minimal concentrations (1-5 mmol/l) changes intensity and direction of hypergastrinemic reaction in the rats. The effect is determined by the anionic composition of salts rather than by the cationic one and depends on the initial concentration of gastrin in blood.     

Ecdysteroids in the haemolymph and the ovaries of the firebrat Thermobia domestica (Packard) (Insecta,Thysanura): Correlations with integumental and ovarian cycles

Ecdysteroids were analysed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) in females of the apterygotous insect Thermobia domestica, which ahs overlapping moulting and reproductive cycles. During each moulting cycle, a peak in the haemolymph concentration of ecdysteroids occurs at day 9 (in the 11-day standard cycle), which can be correlated with apolysis and the beginning of new cuticle deposition. The ovaries show a peak of ecdysteroids at day 5 (i.e. one day before egg-laying), which suggests that these hormones are also involved in the reproductive cycle. In both cases, HPLC analysis combined with RIA suggests that the main ecdysteroid is 20-hydroxyecdysone. This duality in the function of ecdysteroids is discussed.     

Physiological aspects of nm-g mutant: An ecdysteroid-deficient mutant of the silkworm,Bombyx mori

We examined physiological and endocrinological properties of the nm-g mutant of the silkworm, whose development was arrested at 1st or 2nd larval instar. The larva of the nm-g homozygote continued to feed and grow past the stage when a normal larva moulted to the next instar. The nm-g larva attained more than twice the body weight of a normal moulting larva, but remained in the same instar with no sign of the moulting until it finally died. The ecdysteroid concentration in the haemolymph of the nm-g larva was reduced and an increase in the titre, which occurred before moulting in the normal larva, was not observed. Injection of 20-hydroxyecdysone induced the nm-g larva to moult and ecdyse to the next larval instar. From these results, we conclude that nm-g is an ecdysteroid-deficient mutant.     

Determination of Moulting Events in Rock Lobsters from Pleopod Clipping

Rock lobster growth is routinely measured for research to optimise management measures such as size limits and quotas. The process of estimating growth is complicated in crustaceans as growth only occurs when the animal moults. As data are typically collected by tag-recapture methods, the timing of moulting events can bias results. For example, if annual moulting events take place within a very short time-at-large after tagging, or if time-at-large is long and no moulting occurs. Classifying data into cases where moulting has / has not occurred during time-at-large can be required and can generally be determined by change in size between release and recapture. However, in old or slow growth individuals the moult increment can be too small to provide surety that moulting has occurred. A method that has been used since the 1970’s to determine moulting in rock lobsters involves clipping the distal portion of a pleopod so that any regeneration observed at recapture can be used as evidence of a moult. We examined the use of this method in both tank and long-duration field trials within a marine protected area, which provided access to large animals with smaller growth increments. Our results emphasised that determination of moulting by change in size was unreliable with larger lobsters and that pleopod clipping can assist in identifying moulting events. However, regeneration was an unreliable measure of moulting if clipping occurred less than three months before the moult.     

Towards a mechanistic understanding of insect life history evolution: oxygen‐dependent induction of moulting explains moulting sizes

Moults characterise insect growth trajectories, typically following a consistent pattern known as Dyar's rule; proportional size increments remain constant across inter‐instar moults. Empirical work suggests that oxygen limitation triggers moulting. The insect respiratory system, and its oxygen supply capacity, grows primarily at moults. It is hypothesized that the oxygen demand increases with increasing body mass, eventually meeting the oxygen supply capacity at an instar‐specific critical mass where moulting is triggered. Deriving from this hypothesis, we develop a novel mathematical model for moulting and growth in insect larvae. Our mechanistic model has great success in predicting moulting sizes in four butterfly species, indirectly supporting a size‐dependent mechanism underlying moulting. The results demonstrate that an oxygen‐dependent induction of moulting mechanism would be sufficient to explain moulting sizes in the study species. Model predictions deviated slightly from Dyar's rule, the deviations being typically negligible within the present data range. The developmental decisions (e.g. moulting) made by growing larvae significantly affect age and size at maturity, which has important life history implications. The successful modelling of moulting presented here provides a novel framework for the development of realistic insect growth models, which are required for a better understanding of life history evolution.