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.

The distribution and orientation of microtubules were investigated in cells of distinct shapes from different hindgut regions of adult Porcellio scaber Latreille, 1804 and during hindgut morphogenesis in late embryonic and early postembryonic development. All hindgut cells of adult P. scaber contain abundant apico-basal microtubules organized in extensive bundles, but the architecture of bundles is specific for distinct cells. In the anterior chamber the architecture of microtubule bundles closely coincides with different shapes of the cells in this hindgut region and are most prominent in hindgut cells associated with extensive muscles. The shape of cells that form the typhlosole and typhlosole channels is particularly complex. In the papillate region the microtubule bundles protrude between the infoldings of apical plasma membrane and the mitochondria are closely aligned along the microtubules, thus the microtubule bundles in the papillate region are likely involved in the stabilization of the apical labyrinth and positioning of mitochondria. During hindgut morphogenesis the apico-basal microtubule bundles are established relatively late, mainly during early postembryonic development. Morphogenesis of the typhlosole is characterized by coinciding changes in cell shape and microtubule arrangement.

Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control.

To understand developmental processes it is essential to consider both, molecular / genetic data and structural aspects of morphogenesis and differentiation. Integration of both approaches is beneficial to interpret the data obtained and unravel tissue and organism formation. Sequential morphological changes during animal development can be followed by different microscopic methods that enable us to combine imaging of intact embryos, elucidation of the tissue and cell architecture at different size scales, localization of tissue constituents in their original location and correlative imaging, supplemented by analytical microscopy at the specific stages of development. Here we present a combined use of light microscopy, histology, transmission (TEM) and scanning electron microscopy (SEM), labelling with specific ligands and analytical microscopy to unravel the differentiation of epidermis and gut epithelium in a crustacean model. Our study addresses the interrelation of tissue morphogenesis, epithelial cell differentiation at the ultrastructural level and differentiation of the corresponding apical extracellular matrix, the cuticle, examined in the embryos and marsupial larvae of Porcellio scaber. Cuticle is a chitin-based matrix, secreted apically by ectodermal epithelia during development and renewal in arthropods. We integrate structural information at different scale levels with data on matrix elemental and molecular composition in the selected developmental stages. Developmental stages and gross morphological changes in epidermal and gut tissues were identified by imaging the intact embryos and larvae (Fig. 1A), fluorescence labelling of nuclei and imaging of histological sections (Fig. 1B). Architecture of tissues, cells and matrix was characterized at the levels of light and electron microscopy. To determine the organic scaffold composition of the matrix, we performed localization of macromolecules containing N-acetyl-glucosamine (chitin) by labelling with lectin wheat germ agglutinin. The epidermal cuticle in crustaceans is in addition mineralized, forming a stiff exoskeleton. Analytical microscopy was applied to examine calcification of the forming cuticular matrix in marsupial larvae, performed by energy dispersive X-ray spectroscopy (EDS) to analyse elemental composition and Raman spectroscopy to determine mineral forms in the cuticle. The results show that several apical matrices are produced sequentially during epidermis and gut morphogenesis and that matrix renewal is coupled with major morphological modifications such as growth of appendages, elongation or bending of embryo body and hatching from egg envelopes. The early stages of apical matrix formation are similar in the epidermis and gut. Later in marsupial larval stages, specialization of both cuticles is evident, implying the establishment of their specific functions. Exoskeletal cuticle with elaborate surface structures (Fig. 2A) differentiates in several layers displaying chitin-protein fibres patterns (Fig. 2B) and is already prominently calcified. Hindgut cuticle displays differentiation in a homogenous electron lucent procuticle and prominent electron dense epicuticle (Fig. 3A).In concert with the stages of apical matrix formation, a gradual formation of the subapical cell junctions, one of the key factors that determines cell polarity, was evidenced (Fig. 3B). We consider our model system as valuable system to study tissue and cell differentiation in their native environment, with applying the integrated structure-function perspective.

In the fluorescence microscopy of biological specimens the structures in cells and tissues usually need to be labelled with various fluorescent markers. The three main groups of fluorescent markers are small organic fluorochromes, fluorescent proteins and quantum dots. Fluorescent markers differ according to photophysical properties and binding specificity for the selected target structures in the sample. For the labelling of specific structures with small organic fluorochromes or quantum dots it is usually necessary to conjugate them with target-specific macromolecules. For the labelling with fluorescent proteins it is necessary to introduce a fluorescent protein gene into the observed cells or organism. The most important photophysical properties of fluorescent markers are absorption and emission spectra, Stokes shift, extinction coefficient and quantum yield. Currently, various super-resolution fluorescent microscopy techniques exploit special fluorochromes that enable fluorescence modulation by specific wavelength illumination, to achieve the resolution below the diffraction limit.

Ectodermally derived gut and epidermal cells in crustaceans are covered by cuticle, a chitinous apical extracellular matrix, and at the opposite side they are underlined by a basal lamina. Thus, the same cell performs synthesis and spatiotemporal control of elaboration of two extracellular matrices that differ in ultrastructure, composition and functions. Differentiation of the extracellular matrix in epithelial tissues is an integrative part of the establishment and maintenance of the characteristic apicobasal polarized cell architecture. We present here the ultrastructural description of the hindgut cells' apical matrix differentiation during embryonic and larval development of crustacean Porcellio scaber . In addition, in the same developmental stages the ultrastructure of basal lamina was described. The ultrastructural hallmarks of the hindgut cells and their extracellular matrices (ECM) in adults are: (i) apical ECM is a chitinous cuticle, consisting of distinct horizontal layers ‐ electron dense epicuticle and electron lucent procuticle (Fig. 1), (ii) extensive apical and basal plasma membrane infoldings, forming prominent membrane labyrinths, (iii) basal lamina that is forming an extensive interface surface to hemolymph space (Fig. 2). The ultrastructure of cuticle differs in the two anatomic regions of the hindgut, namely the anterior chamber and the papillate region. In the anterior chamber the epicuticle is much thinner than procuticle and lamellae are discernible in the procuticle due to helicoidally arranged chitin protein fibers (Fig. 1). In papillate region epicuticle and procuticle are of approximately equal thicknesses and no lamellae could be discerned in the procuticle. Differentiation of the hindgut cuticular matrix during embryonic and larval development involves a sequential formation and shedding of several apical matrices, which gradually display ultrastructural features characteristic for adults. The outline of the pre‐cuticluar apical matrix in embryos is ruffled and consists of homogenous lucent material and a distal electron dense lamina. In late embryos of stage 18 the first deposition of the apical cuticle was evidenced. In marsupial larvae the cuticle follows the outline of apical hindgut surface and is already differentiated into thin electron dense epicuticle and thicker electron lucent procuticle. In the epicuticle a trilayered surface lamina and electron dense material beneath are discernible (Fig. 3). The procuticle is homogenous along the whole length of the hindgut and non‐lamellate as is characteristic in anterior chamber of adults. The basal side of the hindgut epithelium of marsupial larvae is in contact with basal lamina (Fig. 4) exhibiting fibrillary structure in certain section profiles.