Natural Analogues in pH Variability and Predictability across the Coastal Pacific Estuaries: Extrapolation of the Increased Oyster Dissolution under Increased pH Amplitude and Low Predictability Related to Ocean Acidification.

Coastal-estuarine habitats are rapidly changing due to global climate change, with impacts influenced by the variability of carbonate chemistry conditions. However, our understanding of the responses of ecologically and economically important calcifiers to pH variability and temporal variation is limited, particularly with respect to shell-building processes. We investigated the mechanisms driving biomineralogical and physiological responses in juveniles of introduced (Pacific; Crassostrea gigas) and native (Olympia; Ostrea lurida) oysters under flow-through experimental conditions over a six-week period that simulate current and future conditions: static control and low pH (8.0 and 7.7); low pH with fluctuating (24-h) amplitude (7.7 ± 0.2 and 7.7 ± 0.5); and high-frequency (12-h) fluctuating (8.0 ± 0.2) treatment. The oysters showed physiological tolerance in vital processes, including calcification, respiration, clearance, and survival. However, shell dissolution significantly increased with larger amplitudes of pH variability compared to static pH conditions, attributable to the longer cumulative exposure to lower pH conditions, with the dissolution threshold of pH 7.7 with 0.2 amplitude. Moreover, the high-frequency treatment triggered significantly greater dissolution, likely because of the oyster's inability to respond to the unpredictable frequency of variations. The experimental findings were extrapolated to provide context for conditions existing in several Pacific coastal estuaries, with time series analyses demonstrating unique signatures of pH predictability and variability in these habitats, indicating potentially benefiting effects on fitness in these habitats. These implications are crucial for evaluating the suitability of coastal habitats for aquaculture, adaptation, and carbon dioxide removal strategies.

Exoskeleton dissolution with mechanoreceptor damage in larval Dungeness crab related to severity of present-day ocean acidification vertical gradients.

Ocean acidification (OA) along the US West Coast is intensifying faster than observed in the global ocean. This is particularly true in nearshore regions (<200 m) that experience a lower buffering capacity while at the same time providing important habitats for ecologically and economically significant species. While the literature on the effects of OA from laboratory experiments is voluminous, there is little understanding of present-day OA in-situ effects on marine life. Dungeness crab (Metacarcinus magister) is perennially one of the most valuable commercial and recreational fisheries. We focused on establishing OA-related vulnerability of larval crustacean based on mineralogical and elemental carapace to external and internal carapace dissolution by using a combination of different methods ranging from scanning electron microscopy, energy dispersive X-ray spectroscopy, elemental mapping and X-ray diffraction. By integrating carapace features with the chemical observations and biogeochemical model hindcast, we identify the occurrence of external carapace dissolution related to the steepest Ω calcite gradients (∆Ωcal,60) in the water column. Dissolution features are observed across the carapace, pereopods (legs), and around the calcified areas surrounding neuritic canals of mechanoreceptors. The carapace dissolution is the most extensive in the coastal habitats under prolonged (1-month) long exposure, as demonstrated by the use of the model hindcast. Such dissolution has a potential to destabilize mechanoreceptors with important sensory and behavioral functions, a pathway of sensitivity to OA. Carapace dissolution is negatively related to crab larval width, demonstrating a basis for energetic trade-offs. Using a retrospective prediction from a regression models, we estimate an 8.3% increase in external carapace dissolution over the last two decades and identified a set of affected OA-related sublethal pathways to inform future risk assessment studies of Dungeness crabs.

Structure, function and development of the digestive system in malacostracan crustaceans and adaptation to different lifestyles.

The digestive system of the malacostracan crustaceans, namely the decapods, isopods, amphipods and mysids, is among the most complex organ systems of the animal kingdom serving multiple functions such as food processing, absorption and storage of nutrients, synthesis of digestive enzymes and blood proteins, detoxification of xenobiotics and osmoregulation. It is rather well investigated compared to other invertebrates because the Malacostraca include many ecological keystone species and food items for humans. The Decapoda and Peracarida share food processing with chewing and filtering structures of the stomach but differ with respect to morphology and ultrastructure of the digestive glands. In the Peracarida, the digestive glands are composed of few, relatively large lateral caeca, whereas in the Decapoda, hundreds to thousands of blindly ending tubules form a voluminous hepatopancreas. Morphogenesis and onset of functionality of the digestive system strongly depend on the mode of development. The digestive system is early developed in species with feeding planktonic larvae and appears late in species with direct lecithotrophic development. Some structures of the digestive system like the stomach ossicles are rather constant in higher taxa and are of taxonomic value, whereas others like the chewing structures are to some degree adapted to the feeding strategy. The nutrient absorbing and storing cells of the digestive glands show considerable ultrastructural variation during moult cycle, vitellogenesis and starvation. Some of the various functions of the digestive system are already assigned to specific sections of the digestive tract and cell types, but others still await precise localization.

Ultrastructural differentiation of plasma membrane and cell junctions in the hindgut cells is synchronized with key developmental transitions in Porcellio scaber.

Differentiation of transporting epithelial cells during development of animal organisms includes remodelling of apical and basal plasma membranes to increase the available surface for transport and formation of occluding junctions, which maintain a paracellular diffusion barrier. This study provides a detailed ultrastructural analysis of apical and basal plasma membrane remodelling and cell junction formation in hindgut cells during late embryonic and early postembryonic development of the crustacean Porcellio scaber. Hindgut cells in late-stage embryos are columnar with flat apical and basal plasma membranes. In early-stage marsupial mancae the hindgut cells begin to acquire their characteristic dome shape, the first apical membrane folding is evident and the septate junctions expand considerably, all changes being probably associated with the onset of active feeding. In postmarsupial mancae the apical labyrinth is further elaborated and the septate junctions are expanded. This coincides with the transition to an external environment and food sources. First basal infoldings appear in the anterior chamber of early-stage marsupial mancae, but in the papillate region they are mostly formed in postmarsupial mancae. In molting late-stage marsupial mancae, the plasma membrane acquires a topology characteristic of cuticle-producing arthropod epithelia and the septate junctions are considerably reduced.

Intra-annual dynamics of phloem formation and ultrastructural changes in sieve tubes in Fagus sylvatica.

Despite increased interest in the timing and dynamics of phloem formation, seasonal changes in the structure of phloem sieve elements remain largely unexplored. To understand better the dynamics of phloem formation and the functioning of sieve tubes in the youngest phloem in Fagus sylvatica L., we investigated repeatedly taken phloem samples during the growing season of 2017 by means of light microscopy, and transmission and scanning electron microscopy. Phloem formation started with the expansion of the overwintered early phloem sieve tubes adjacent to the cambium and concurrent cambial cell production. The highest phloem growth rate was observed in general 1 week after the onset of cambial cell production, whereas the transition from early to late phloem occurred at the end of May. Cambial cell production ceased at the end of July. The final width of the phloem increment was 184 ± 10 µm, with an early phloem proportion of 59%. Collapse of older phloem tissue is a progressive process, which continuously occurred during the sampling period. Collapse of early phloem sieve tubes started shortly after the cessation of cambial cell production. Prior to the onset of radial growth, late phloem from the previous year represented 80% of the total non-collapsed part; during the growth period, this percentage decreased to 20%. Differences were observed in both sieve tube ultrastructure and sieve plate geometry between the youngest and older phloem. However, sieve plates were never completely occluded by callose, suggesting that processes affecting the functionality of sieve tubes may differ in the case of regular collapse or injury. The youngest parts of the phloem increment from the previous year (i.e., previous late phloem) continue functioning for some time in the current growing season, but the two-step development of overwintered phloem cells also ensures a sufficient translocation pathway for photosynthates to the actively growing tissues.

Comparative ultrastructure of cells and cuticle in the anterior chamber and papillate region of Porcellioscaber (Crustacea, Isopoda) hindgut.

Isopod hindgut consists of two anatomical and functional parts, the anterior chamber, and the papillate region. This study provides a detailed ultrastructural comparison of epithelial cells in the anterior chamber and the papillate region with focus on cuticle ultrastructure, apical and basal plasma membrane labyrinths, and cell junctions. Na+/K+-ATPase activity in the hindgut epithelial cells was demonstrated by cytochemical localisation. The main difference in cuticle ultrastructure is in the thickness of epicuticle which is almost as thick as the procuticle in the papillate region and only about one sixth of the thickness of procuticle in the anterior chamber. The apical plasma membrane in both hindgut regions forms an apical plasma membrane labyrinth of cytoplasmic strands and extracellular spaces. In the papillate region the membranous infoldings are deeper and the extracellular spaces are wider. The basal plasma membrane is extensively infolded and associated with numerous mitochondria in the papillate region, while it forms relatively scarce basal infoldings in the anterior chamber. The junctional complex in both hindgut regions consists of adherens and septate junctions. Septate junctions are more extensive in the papillate region. Na+/K+-ATPase was located mostly in the apical plasma membranes in both hindgut regions. The ultrastructural features of hindgut cuticle are discussed in comparison to exoskeletal cuticle and to cuticles of other arthropod transporting epithelia from the perspective of their mechanical properties and permeability. The morphology of apical and basal plasma membranes and localisation of Na+/K+-ATPase are compared with other arthropod-transporting epithelia according to different functions of the anterior chamber and the papillate region.

Structural optimization and amorphous calcium phosphate mineralization in sensory setae of a terrestrial crustacean (Isopoda: Oniscidea).

Terrestrial isopods possess large sensory setae on their walking legs. Increased fracture resistance of these elongated structures is of crucial importance, making the exoskeleton forming the setae an interesting durable material that may inspire biomimetic designs. We studied the cuticle of the sensory setae with analytical electron microscopy in order to gain detailed insights into its structure and composition at the nanometer scale and identify features that increase the fracture resistance of these minute skeletal elements. The setae are stiff structures formed by mineralized cuticle that are connected to the leg exoskeleton by a non-mineralized joint membrane. Our results demonstrate that different layers of the setal cuticle display contrasting organizations of the chitin-protein fibers and mineral particles. While in the externally positioned exocuticle organic fibers shift their orientation helicoidally in sequential layers, the fibers are aligned axially in the internally positioned endocuticle. In the setal cuticle, layers of structurally anisotropic cuticle likely providing strength in the axial direction are combined with layers of isotropic cuticle which may allow the setae to better resist perpendicular loading. They are further strengthened with amorphous calcium phosphate, a highly fracture resistant mineral rarely observed in invertebrate skeletons.

Development of calcium bodies in Hylonsicus riparius (Crustacea: Isopoda).

Calcium bodies are internal epithelial sacs found in terrestrial isopods of the family Trichoniscidae that contain a mineralized extracellular matrix that is deposited and resorbed in relation to the molt cycle. Calcium bodies in several trichoniscids are filled with bacteria, the function of which is currently unknown. The woodlouse Hyloniscus riparius differs from other trichoniscids in that it possesses two different pairs of calcium bodies, the posterior pair being filled with bacteria and the anterior pair being devoid of bacteria. We explored the development of these organs and bacterial colonization of their lumen during the postmarsupial development with the use of optical clearing and whole-body confocal imaging of larval and juvenile stages. Our results show that calcium bodies are formed as invaginations of the epidermis in the region of intersegmental membranes during the postmarsupial development. The anterior pair of calcium bodies is generated during the first postmarsupial manca stage, whereas the posterior calcium bodies first appear in juveniles and are immediately colonized by bacteria, likely through a connection between the calcium body lumen and the body surface. Mineral is deposited in calcium bodies as soon as they are present.

Polyphosphate-accumulating bacterial community colonizing the calcium bodies of terrestrial isopod crustaceans Titanethes albus and Hyloniscus riparius.

Terrestrial isopods from the group Trichoniscidae accumulate calcium in specialized organs, known as the calcium bodies. These consist of two pairs of epithelial sacs located alongside the digestive system. These organs contain various forms of calcium and constantly present bacteria. To elucidate their origin and role, we analyzed the bacteria of the calcium bodies in the cave-dwelling isopod Titanethes albus and the epigean species Hyloniscus riparius, by microscopy, histochemistry, energy dispersive X-ray spectrometry, 16S rRNA analysis and in situ hybridization. The calcium bodies of both species comprise numerous and diverse bacterial communities consisting of known soil bacteria. Despite their diversity, these bacteria share the polyphosphate-accumulation ability. We present the model of phosphorous dynamics in the calcium bodies during the molting cycle and potentially beneficial utilization of the symbiotic phosphate by the host in cyclic regeneration of the cuticle. Although not fully understood, this unique symbiosis represents the first evidence of polyphosphate-accumulating bacterial symbionts in the tissue of a terrestrial animal.

Cuticle morphogenesis in crustacean embryonic and postembryonic stages.

The crustacean cuticle is a chitin-based extracellular matrix, produced in general by epidermal cells and ectodermally derived epithelial cells of the digestive tract. Cuticle morphogenesis is an integrative part of embryonic and postembryonic development and it was studied in several groups of crustaceans, but mainly with a focus on one selected aspect of morphogenesis. Early studies were focused mainly on in vivo or histological observations of embryonic or larval molt cycles and more recently, some ultrastructural studies of the cuticle differentiation during development were performed. The aim of this paper is to review data on exoskeletal and gut cuticle formation during embryonic and postembryonic development in crustaceans, obtained in different developmental stages of different species and to bring together and discuss different aspects of cuticle morphogenesis, namely data on the morphology, ultrastructure, composition, connections to muscles and molt cycles in relation to cuticle differentiation. Based on the comparative evaluation of microscopic analyses of cuticle in crustacean embryonic and postembryonic stages, common principles of cuticle morphogenesis during development are discussed. Additional studies are suggested to further clarify this topic and to connect the new knowledge to related fields.

Exoskeletal cuticle of cavernicolous and epigean terrestrial isopods: A review and perspectives.

Comparative ultrastructural studies of the integument in terrestrial isopod crustaceans show that specific environmental adaptations of different eco-morphotypes are reflected in cuticle structure. The biphasic molting in isopods is a valuable experimental model for studies of cuticular matrix secretion and degradation in the same animal. The aim of this review is to show structural and functional adaptations of the tergal cuticle in terrestrial isopods inhabiting cave habitats. Exoskeletal cuticle thickness, the number of cuticular layers, epicuticle structure, mineralization, pigmentation and complexity of sensory structures are compared, with greater focus on the well-studied cave trichoniscid Titanethes albus. A large number of thinner cuticular layers in cave isopods compared to fewer thicker cuticular layers in related epigean species of similar body-sizes is explained as a specific adaptation to the cavernicolous life style. The epicuticle structure and composition are compared in relation to their potential waterproofing capacity in different environments. Cuticle mineralization is described from the functional point of view as well as from the aspect of different calcium storage sites and calcium dynamics during the molt cycle. We also discuss the nature and reduction of pigmentation in the cave environment and outline perspectives for future research.

Axially aligned organic fibers and amorphous calcium phosphate form the claws of a terrestrial isopod (Crustacea).

Skeletal elements that are exposed to heavy mechanical loads may provide important insights into the evolutionary solutions to mechanical challenges. We analyzed the microscopic architecture of dactylus claws in the woodlice Porcellio scaber and correlated these observations with analyses of the claws' mineral composition with energy dispersive X-ray spectrometry (EDX), electron energy loss spectroscopy (EELS) and selected area electron diffraction (SAED). Extraordinarily, amorphous calcium phosphate is the predominant mineral in the claw endocuticle. Unlike the strongly calcified exocuticle of the dactylus base, the claw exocuticle is devoid of mineral and is highly brominated. The architecture of the dactylus claw cuticle is drastically different from that of other parts of the exoskeleton. In contrast to the quasi-isotropic structure with chitin-protein fibers oriented in multiple directions, characteristic of the arthropod exoskeleton, the chitin-protein fibers and mineral components in the endocuticle of P. scaber claws are exclusively axially oriented. Taken together, these characteristics suggest that the claw cuticle is highly structurally anisotropic and fracture resistant and can be explained as adaptations to predominant axial loading of the thin, elongated claws. The nanoscale architecture of the isopod claw may inspire technological solutions in the design of durable machine elements subjected to heavy loading and wear.

Quantitative Morphology of Epithelial Folds.

The shape of spatially modulated epithelial morphologies such as villi and crypts is usually associated with the epithelium-stroma area mismatch leading to buckling. We propose an alternative mechanical model based on intraepithelial stresses generated by differential tensions of apical, lateral, and basal sides of cells as well as on the elasticity of the basement membrane. We use it to theoretically study longitudinal folds in simple epithelia and we identify four types of corrugated morphologies: compact, invaginated, evaginated, and wavy. The obtained tissue contours and thickness profiles are compared to epithelial folds observed in invertebrates and vertebrates, and for most samples, the agreement is within the estimated experimental error. Our model establishes the groove-crest modulation of tissue thickness as a morphometric parameter that can, together with the curvature profile, be used to estimate the relative differential apicobasal tension in the epithelium.

Annual Cambial Rhythm in Pinus halepensis and Pinus sylvestris as Indicator for Climate Adaptation.

To understand better the adaptation strategies of intra-annual radial growth in Pinus halepensis and Pinus sylvestris to local environmental conditions, we examined the seasonal rhythm of cambial activity and cell differentiation at tissue and cellular levels. Two contrasting sites differing in temperature and amount of precipitation were selected for each species, one typical for their growth and the other represented border climatic conditions, where the two species coexisted. Mature P. halepensis trees from Mediterranean (Spain) and sub-Mediterranean (Slovenia) sites, and P. sylvestris from sub-Mediterranean (Slovenia) and temperate (Slovenia) sites were selected. Repeated sampling was performed throughout the year and samples were prepared for examination with light and transmission electron microscopes. We hypothesized that cambial rhythm in trees growing at the sub-Mediterranean site where the two species co-exist will be similar as at typical sites for their growth. Cambium in P. halepensis at the Mediterranean site was active throughout the year and was never truly dormant, whereas at the sub-Mediterranean site it appeared to be dormant during the winter months. In contrast, cambium in P. sylvestris was clearly dormant at both sub-Mediterranean and temperate sites, although the dormant period seemed to be significantly longer at the temperate site. Thus, the hypothesis was only partly confirmed. Different cambial and cell differentiation rhythms of the two species at the site where both species co-exist and typical sites for their growth indicate their high but different adaptation strategies in terms of adjustment of radial growth to environmental heterogeneity, crucial for long-term tree performance and survival.

Size fractionation and size characterization of nanoemulsions of lipid droplets and large unilamellar lipid vesicles by asymmetric-flow field-flow fractionation/multi-angle light scattering and dynamic light scattering.

Asymmetric-flow field-flow fractionation technique coupled to a multi-angle light-scattering detector (AF4-MALS) was used together with dynamic light-scattering (DLS) in batch mode and transmission electron microscopy (TEM) to study the size characteristics of the trioleoylglycerol lipid droplets covered by a monolayer of sphingomyelin and cholesterol, in water phase. These lipid droplet nanoemulsions (LD) were formed by ultrasonication. In parallel, the size characteristics of large unilamellar lipid vesicles (LUV) prepared by extrusion and composed of sphingomyelin and cholesterol were determined. LD and LUV were prepared at two different molar ratios (1/1, 4/1) of sphingomyelin and cholesterol. In AF4-MALS, various cross-flow conditions and mobile phase compositions were tested to optimize the separation of LD or LUV particles. The particle radii, R, as well as the root-mean-square radii, Rrms, of LD and LUV were determined by AF4-MALS, whereas the hydrodynamic radii, Rh, were obtained by DLS. TEM visualization revealed round shape particles of LD and LUV.

Formation of the hindgut cuticular lining during embryonic development of Porcellioscaber (Crustacea, Isopoda).

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

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda).

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.

Clinical and structural features of sperm head vacuoles in men included in the in vitro fertilization programme.

The human sperm head vacuoles and their role in male infertility are still poorly understood. The aim of this study was to identify the clinical and ultrastructural features of human sperm head vacuoles in men included in the in vitro fertilization programme: men with normal (normozoospermia) and impaired sperm morphology (teratozoospermia). The sperm samples were observed under 6000-time magnification using motile sperm organelle morphology examination (MSOME). The proportion of sperm with head vacuoles was evaluated and related to the outcome of in vitro fertilization. The sperm of men with impaired sperm morphology was characterized by a higher proportion of sperm head vacuoles. The sperm head vacuoles were related to impaired semen quality (sperm concentration, motility, and morphology) but were not influenced by male factors (semen volume, height, age, weight, or body mass index). Moreover, sperm head vacuoles were related to impaired fertilization rate merely after classical in vitro fertilization (IVF), while there was no relation to pregnancy. In a subgroup of men, the sperm was fixed and observed by transmission electron microscopy (TEM). The ultrastructural study revealed that sperm head vacuoles are large nuclear indentations of various sizes and positions, packed with membranous material organized in membrane whorls (MW).

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.

Eco-morphological studies on pleopodal lungs and cuticle in Armadillidium species (Crustacea, Isopoda, Oniscidea).

Terrestrial isopods (Crustacea, Isopoda, Oniscidea) have adapted to land life by diverse morphological, physiological and behavioral changes. Woodlice species exhibit a large variety in this respect, their preferences ranging from moist to dry habitats. These moisture preference values are related to various morphological adaptations, rendering terrestrial isopods amenable to studying morphological adaptations to terrestrial life. We performed a comparison of four Armadillidium species (Armadillidium zenckeri, Armadillidium nasatum, Armadillidium versicolor, Armadillidium vulgare), by quantifying two morphological traits: the extent of the interfacial endothelium between the respiratory space and the hemolymph within pleopodal lungs and the thickness of tergite cuticle, which are 'key factors' in determining protection from desiccation. These values were measured from light micrographs of cross-sectioned lungs. The cosmopolitan A. vulgare, as a habitat generalist, seems to be the most resistant against desiccation and other environmental conditions, while A. zenckeri is the most sensitive one. Light microscopic studies revealed that the four species can be ordered similarly, if we compare them by the extension of the endothelial interface and cuticle thickness, suggesting that these morphological traits are important determinants of their distribution on habitat, microhabitat scales and through the existence of suitable habitats - together with many other factors - the geographical pattern of species occurence.