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.     

Ultrastructure and mineral distribution in the tergal cuticle of the terrestrial isopod Titanethes albus. Adaptations to a karst cave biotope

Composition and spatial distribution of organic and inorganic materials within the cuticle of isopods vary between species. These variations are related to the behaviour and habitat of the animal. The troglobiotic isopod Titanethes albus lives in the complete darkness of caves in the Slovenian Karst. This habitat provides constant temperature and saturated humidity throughout the year and inconsistent food supply. These conditions should have lead to functional adaptations of arthropod cuticles. However, studies on structure and composition of cave arthropod cuticles are rare and lacking for terrestrial isopods. We therefore analysed the tergite cuticle of T. albus using transmission and field-emission electron microscopy, confocal μ-Raman spectroscopic imaging, quantitative X-ray diffractometry, thermogravimetric analysis and atomic absorption spectroscopy. The ultrastructure of the epicuticle suggests a poor resistance against water loss. A weak interconnection between the organic and mineral phase within the endo- and exocuticle, a comparatively thin apical calcite layer, and almost lack of magnesium within the calcite crystal lattice suggest that the mechanical strength of the cuticle is low in the cave isopod. This may possibly be of advantage in maintaining high cuticle flexibility and reducing metabolic expenditures.     

Calcium translocations during the moulting cycle of the semiterrestrial isopod <Emphasis Type="Italic">Ligia hawaiiensis</Emphasis> (Oniscidea,Crustacea)

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO₃ deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. ⁴⁵Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.     

Structural investigation of the female genitalia and sperm-storage sites in the terrestrial isopod Armadillidium vulgare (Crustacea, Isopoda)

The cuticular genitalia of the terrestrial isopod, Armadillidium vulgare, have two distinct states during the reproductive cycle of the females. The structural differences between the reproductive and non-reproductive states, and the structure of the sperm storage sites were investigated employing electron and light microscopy. In both states the genitalia consist of a distal segment that connects to the gonopore, and a cuticular tube-like structure lining the lumen of the oviduct in the middle region of the oviduct. Sheath-like projections, apparently consisting of cuticular material, extend laterally along two sides of the cuticular tube. In the proximal region of the oviduct cuticular structures are lacking. In the non-reproductive state the distal segment consists of endo-, exo- and epicuticle. The exocuticle is three layered with unusual spongy and dense layers at the distal side. On one side the endocuticle doubles in thickness to form a cuticular bulge that fills the lumen of the distal segment leaving just a narrow U-shaped space. The cuticular tube consists of endo- and epicuticle only. In the reproductive state the distal segment is funnel-shaped and forms branched cuticular folds that increase in complexity from distal to proximal. In the cuticular tube these folds tightly fill the lumen of the oviduct. At the confluence of the oviduct with the ovary spermatozoa are stored in a seminal receptacle.     

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.     

Ultrastructure and formation of the physogastric termite queen cuticle

The physogastric termite queen is the most striking example in insects of growth in size without cuticular moulting. This phenomenon has been studied with electron microscopy and histochemical tests in two species of higher termites, Cubitermes fungifaber and Macrotermes bellicosus. The abdominal hypertrophy (physogastry) is allowed by growth of the arthrodial membranes of the swarming imago. The growth is slow (over several years) but important: the cuticular dry weight is multiplied by 20 in C. fungifaber, by 100-150 in M. bellicosus. The termite queen cuticle arises from the transformation of the cuticle of the swarming imago or imaginal cuticle (unfolding and growing of the epicuticle, stretching of the endocuticle, resorption of the subcuticle) and from the secretion of a new endocuticle or royal endocuticle. The termite queen is the first example known in insects of epicuticular growth. In the physogastric queen, three cuticular types are observed: the rigid cuticle of the sclerites, the soft cuticle of the arthrodial membranes and the partially rigid cuticle of special structures, the neosclerites, which show both rigidity and growth. The fibrillar architecture varies according to the abdominal zones and the position within the cuticle. It appears to be determined by the forces arising from the musculature and the anisometric abdominal growth. The king does not become physogastric, although its cuticle is also modified.     

The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis

The insoluble matrix of larval shells of the marine bivalve mollusk Mytilus galloprovincialis is investigated by confocal laser scanning microscopy using a GFP fusion protein with a chitin-binding domain for labeling of chitinous structures. We show that chitinous material is present in the larval shell, presumably as a chitin-protein complex. We further show that the structure of the chitinous material changes with the development of the larvae. We conclude from the presence of characteristic chitinous structures in certain shell regions that chitin fulfills an important function in the formation and functionality of larval bivalve shells.     

Evolutionary biogeography of the Microparasellid isopod Microcharon (Crustacea) in the Mediterranean Basin

The interstitial subterranean isopod Microcharon (Crustacea) is highly diversified within the Mediterranean. The present distribution of 70 species is considered the result of the joint history of this genus and its environment. This stygobiont is derived from marine surface ancestors. Both Plate Tectonics and the two-step model of colonization and evolution, the second phase of which represents vicariance during the Tethys regressions, enable an understanding of the evolutionary history of this monophyletic group. The single extant marine littoral species was separated from its sister group by the closure of the Gibraltar Straits during the Messinian. The drift of the Apulian plate, the Alboran-Kabylian-Calabrian fragment as well as the opening of the western Mediterranean through the translation of the Corsican-Sardinia plate could explain the divergences in the western and the eastern Mediterranean. The primitive sister groups in Morocco and in Spain would have been left by the Turonian Tethys regression. Such regressions of the Tethys embayments could have played a major role in the evolution of the genus from Turanian up to Tortonian in Norht Africa, and up to Pliocene and even to Pleistocene in Italian, Iberian and Greek zones. Several eastern European species may be a product of the Paratethys regressions. Everywhere, species with plesiomorphic characters are related to old regressions and vice versa. Local geological events could have resulted in further vicariance divergences. There is a good congruence between the evolutionary history of Microcharon and other stygobiont isopod, amphipods or syncarids living in the same regions studied in the Mediterranean.Presented at the Symposium of Biogeography at Subterranean Crustaceans: the effects of different scales (June 1992; Charleston, South Carolina, U.S.A.). Manuscrips prepared for print by J. R. Holsinger.     

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.     

Neurotransmitters of motor neurons in the stomatogastric ganglion of an isopod crustacean, Ligia exotica

Neurotransmitters of motor neurons in the foregut muscles of an isopod Ligia exotica were identified by recording changes in membrane potential to exogenously applied glutamate and acetylcholine. The effects of antagonists, tubocurare and joro spider toxin, on excitatory junctional potentials evoked by nerve stimulation and by iontophoretic application of glutamate and acetylcholine provided additional evidence for identification. The junctional receptors were desensitized by putative neurotransmitters. Glutamate is a candidate as an excitatory neurotransmitter at the neuromuscular junctions in intrinsic muscles of the gastric mill and pylorus, and acetylcholine is a candidate in the extrinsic muscles of the gastric mill and cardiopyloric valve.     

Ultrastructure of cuticular exudations in parasitic juvenileHeterodera schachtii,as related to cuticle structure

Summary The development ofHeterodera schachtii inside roots of a cruciferous host plant grown under monoxenic conditions in an agar medium was observed with video-enhanced contrast light microscopy. One to 6 days after inoculation, roots were excised and processed for electron microscopic observations. Exudates were present on the cuticle surfaces of J 2 and early J 3 juveniles located at feeding sites. Fibrillar exudations were correlated with similar fibrillar patterns in the epicuticle, exocuticle, intermediate zone, and the striated endocuticle. Secretion vesicles assembled at many Golgi sites in the hypodermis, appeared to coalesce and form large electron translucent vesicles in the cytoplasm. We propose that secretion vesicles migrate toward the cuticle, contact the plasmalemma and transfer their contents by exocytosis or a similar mechanism to a secretion accumulation site. These contents are associated with cuticle structure and emerge as surface exudations.     

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.     

Composition and distribution of benthic isopod (Crustacea,Malacostraca) families off the Victoria-Land Coast (Ross Sea,Antarctica)

The benthic fauna off the Victoria-Land-Coast, Ross Sea (Antarctica) was investigated during the 19th Italica expedition in February 2004. Samples were taken along a latitudinal transect from Cape Adare down to Terra Nova Bay at water depths ranging from 84 to 515 m. A Rauschert dredge was used at 18 stations to collect epi- and infaunal macrobenthos. 9,494 specimens of Isopoda were collected, representing 19 families. Desmosomatidae were the most abundant family (35,297 ind/1,000 m²), followed by Paramunnidae (23,973 ind/1,000 m²). Paramunnidae was the most frequent taxon and was collected at all stations, in contrast to the Desmosomatidae, which did not occur at any station off Cape Adare.     

Changes in osmotic and ionic concentrations in the hemolymph of Macrobrachium rosenbergii exposed to varying salinities and correlation to ionic and crystalline composition of the cuticle

Osmotic and ionic regulatory ability were examined in the giant freshwater prawn, Macrobrachium rosenbergii in response to varying salinities. In freshwater, and under conditions of low salinity, hemolymph osmolality was maintained around 450 mOsm. Under high salinity, osmolality values increased in a time-wise manner until reaching levels of the surrounding rearing water. Changes in sodium concentration generally paralleled osmotic change, and potassium and magnesium concentrations increased upon exposure to extremely high salinity. In contrast, total calcium concentration was maintained at high levels regardless of salinity treatment. Examination of crystalline structure and ionic composition of the cuticle revealed that it was comprised principally of an α-chitin-like material, and calcite (calcium carbonate). Calcite accounted for 25% of total bulk weight in freshwater, while sodium, potassium and magnesium constituents combined comprised less than 2.5% of this total. Although sodium, potassium and magnesium contents increased nearly 2-fold in response to changing salinity, calcium levels remained relatively constant.     

Morphology and evolution of respiratory structures in the pleopod exopodites of terrestrial Isopoda (Crustacea, Isopoda, Oniscidea)

The morphology of the respiratory structures located in the pleopod exopodites of terrestrial Isopoda is described for representative species of different genera. Special emphasis is set on the evolution of these lungs in the context of phylogenetic relationships as revealed by other morphological characters. In the most primitive species of the Oniscidea, and still of subordinate taxa of the Crinocheta, respiration takes place in the thin ventral integument of the exopodites. The evolution of dorsal respiratory fields with a weakly wrinkled surface occurred at least six times within the Crinocheta. In five of these cases, a further development can be observed. The evolution of a partly covered respiratory field with strongly wrinkled surface may have taken place six times. It is assumed that completely internalized lungs with spiracles surrounded by a water‐repellent surface microsculpture, evolved at least six times independently within the Oniscidea: in the Tylidae, Actaecia, Aphiloscia, the Eubelidae, the Armadillidae and in a taxon probably comprising Porcellionidae plus Armadillidiidae.     

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.     

Ultrastructure and distribution of identified neurosecretory terminals in the sinus gland of the terrestrial isopod Oniscus asellus

An ultrastructural study of the sinus gland of the terrestrial isopod, Oniscus asellus, reveals that this structure consists of two regions: the bulb, which is attached by a narrow stalk to the optic lobe, and the lateral extension, which extends from the bulb along the optic tract to the compound eye. The bulb has a distal region containing only neurosecretory terminals, and a proximal region containing terminals, glial cells, and axons that give rise to the distally located terminals. In total, the sinus gland contains five types of terminals which can be distinguished by their location and the appearance of their neurosecretory granules. Three terminal types are located in the bulb and two in the lateral extension. The size of the terminals in the bulb varies among the three types, but the number of terminals is approximately the same for each type. Conversely, the two terminal types in the lateral extension are similar in size, but differ in number. Axons of two terminal types in the bulb can be traced to the central region of the protocerebrum; axons of one terminal type in the bulb and of terminals in the lateral extension can be traced to the optic lobe.     

Ultrastructural changes in the cuticle of the sheep blowfly, Lucilia, induced by certain insecticides and biological inhibitors

The ultrastructural effects on larval cuticle of Lucilia cuprina of two inhibitors of chitin synthesis, diflubenzuron and polyoxin D and an inhibitor of dihydrofolate reductase, aminopterin, are compared with those of the insecticide, cyromazine. Diflubenzuron and polyoxin D both prevent the formation of a normal lamellate appearance in procuticle and interfere with deposition of epicuticle. Aminopterin and cyromazine cause necrotic lesions in the cuticle which, in the case of cyromazine, are contiguous with invasive processes of epidermal cells. There is an accumulation of electron-dense granules in some epidermal cells in larvae poisoned with aminopterin or cyromazine. Aminopterin has a more drastic cytotoxic effect than cyromazine and it also interferes with the formation of epicuticle. The lesions produced by cyromazine treatment are not mimicked precisely by any of the other chemicals. However, there is closer accord between the effects of cyromazine and aminopterin than between cyromazine and the inhibitors of chitin formation.     

Comparative ultrastructural study of the cuticle and spermatozoa in Propappus volki Michaelsen, 1916 (Annelida: Clitellata)

The ultrastructure of the cuticle and mature spermatozoa of the oligochaete Propappus volki Michaelsen, 1916 is described with the aim of providing additional data for clarifying the systematic position of the taxon. P. volki is a fresh-water species living in streams, and is easily recognized by its proboscis on the pre-segmental prostomium and, in mature specimens, by a clitellum covering the segments XII–XIV. The cuticle is composed of a proximal fibre zone and a distal layered epicuticle covered with membrane-bound epicuticular projections. The fibre zone consists of collagenous fibres in a matrix, arranged in either densely packed parallel layers with the fibres oriented in the same direction, or with more loosely distributed fibres, although with the same main orientation. The epicuticular projections are pyramidal with the base leaning on the outer surface of the epicuticle. The cuticle covering the proboscis differs in morphology from that of the rest of the worm; the fibre zone is composed of thin and short fibrils running in all directions, and the epicuticular projections are longer and more narrow than the projections in other regions of the worm.

The spermatozoa are filiform cells formed, in sequence, by an acrosome, an elongated nucleus, a long midpiece, and a flagellum. The acrosomal tube is short and straight with a completely external acrosomal vesicle. Following the acrosome is a apically corkscrew-shaped and basally straight nucleus. The midpiece is twisted and formed by five mitochondria. The flagellum shows a prominent central sheath arrangement.

A comparison with ultrastructurally described cuticles and spermatozoa from other clitellate species reveals most similarities with enchytraeids.