Changes in the avascular area of the meniscus using mesenchymal stem cells and growth plate chondrocytes in a pig model.

Menisci are wedge-shaped cartilage discs that are divided into two parts: the avascular and vascular regions. They are formed by fibrocartilage tissue, which contains round cartilage-like cells and extracellular matrix. Meniscus injury in animals is a common orthopedic problem, but data on the natural healing process mainly deals with the vascular zone. The healing processes in the avascular zone of the meniscus are significantly limited. Thus, this study aimed to evaluate autologous growth plate chondrocytes' impact on the healing process of a damaged meniscus in the avascular zone based on a growing animal model. The study group consisted of 10 pigs at about three months of age. From each animal, chondrocytes from the iliac growth plate and from concentrated bone marrow were taken. Knee joints were divided into right (R) and left (L). The medial meniscus of the R knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates (nCHON). The medial meniscus of the L knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates supplemented with immature chondrocytes isolated from growth plates (wCHON). The meniscus was damaged in the avascular zone in both knee joints. Followingly, the damaged part of the meniscus was filled with a scaffold with cells from the concentrated bone marrow and from growth plate chondrocytes. In the control group, a scaffold with concentrated bone marrow cells was used. After three months the animals were euthanized and preparations (microscopic slides) were made from the meniscus' damaged part. A qualitative and quantitative analysis have been prepared. The wCHON group in comparison with the nCHON group showed a statistically significantly higher number of fusiform cells on the surface of the graft as well as better healing of the graft. In addition, the degree of vascularization was higher in specimens from the wCHON group than in the nCHON group. The results of our research on immature pig knees revealed that mesenchymal stem cell and growth plate chondrocytes could be treated as the cell source for meniscus reconstruction, and growth plate chondrocytes enhance healing processes in the avascular zone of the injured meniscus.

Microevolution or wide tolerance? Level of stress proteins in the beet armyworm Spodoptera eqigua hübner (Lepidoptera: Noctuidae) exposed to cadmium for over 150 generations.

The aim of this study was to investigate whether the cadmium tolerance developed in the beet armyworm Spodoptera exigua selected for over 150 generations may be related to synthesis of the stress proteins metallothioneins (Mts) and 70 kDa heat shock proteins (HSP70). To achieve this, six S. exigua strains (control, k), 150-generation Cd exposure strain (cd), and four 18-generation Cd exposure strains differing in Cd concentration (cd44, cd22, cd11, cd5) were reared. Stress protein level was measured in the midgut of the 5th larval stage after 1-6, 12 and 18 generations. Cd contents was measured in the pupae. Unlike Cd concentration, which depended on metal contents in food but was not generation-dependent, the pattern of Mts and HSP70 concentrations changed in experimental strains from generation to generation. Stress protein levels in the insects exposed to the highest Cd concentration (the same as in the 150-generation Cd exposure strain), initially higher than in the control strain, after the 12th generation did not differ from the level measured in the control strains. It seems therefore that stress proteins play a protective role in insects of lower tolerance to cadmium. The tolerance developed during multigenerational exposure probably relies on mechanisms other than Mt and HSP70 synthesis.

Changes in the midgut cells in the European cave spider, Meta menardi, during starvation in spring and autumn.

During the growth period, in surface habitats, spiders catch enough prey to feed normally. In contrast, in the cave entrance zone, prey may be relatively scarce. Meta menardi inhabits this cave section, resulting in temporary starvation. We studied structural changes in the midgut epithelial cells of M. menardi during a short-term and a medium-term controlled starvation to mimic the occasional starvation in caves, during spring and autumn. Digestive cells, secretory cells and adipocytes were examined before the experimental starvation, in the middle and at the end of starvation. We used light microscopy, transmission electron microscopy and specific histochemical methods for the detection of lipids, polysaccharides and proteins. Detection of lysosomes, autolysosomes and apoptosis was also carried out. The general structures of the cells did not change during the experimental starvation in either season, while some specific differences in the ultrastructure were observed. In both sexes, in both seasons, the amounts of lipids, glycogen and proteins decreased during starvation. Larger amounts of lipids were found in autumn, while there were no significant differences in the amounts of glycogen and proteins. In both sexes, in both seasons, autophagy and apoptosis intensified with starvation in progress, but more intensively in females. Thus, autumn individuals, in contrast to spring ones, compile energy-supplying stores to confront the subsequent winter deficiency of prey in caves, while the cellular ultrastructures undergo the same starvation-dependant changes at any time during the growth period.

Effects of food contaminated with cadmium and copper on hemocytes of Steatoda grossa (Araneae: Theridiidae).

The aim of this study was to evaluate the metabolic condition of Steatoda grossa (Theridiidae) spider, from their hemocytes, after a short-term (four-week) exposure to cadmium and copper in sublethal doses by administering them into the body of the preys. The ultrastructure of the dominant types of hemocytes, such as granulocytes, plasmatocytes and prohemocytes, was evaluated using transmission electron microscope (TEM). Quantitative evaluation of apoptotic and necrotic cells, as well as the ones with depolarized mitochondria in hemolymph, was performed using flow cytometry, while ATP concentration and ADP/ATP ratio in hemocytes were measured by luminescent methods. Cadmium, unlike copper, demonstrated proapoptotic and pronecrotic activity. Low ATP levels and high ADP/ATP ratio in hemocytes indicate a disturbance in the energy metabolism of cells and may account for their qualitative and quantitative degenerative changes. The intensification of death processes in hemocytes after an exposure to cadmium-contaminated food may impair the ability of these cells to fight infectious diseases. Copper at the applied dosage was safe for the spiders without causing visible changes in the hemocyte ultrastructure and in the level of analyzed cell death indices.

Oxidative stress and genotoxic effects of diamond nanoparticles.

Due to the unique and useful properties of nanodiamonds (ND), their production and use is rapidly increasing. Thus, more of these particles will be released into the environment and organisms will inevitably be exposed to them. The current knowledge about the toxicity of ND, especially in vivo toxicity, is fragmentary. In this study, the toxicity of nanodiamonds was assessed in Acheta domesticus following chronic exposure to different nominal concentrations of ND (20 and 200µgg(-1) food) administrated in food for the entire lifespan. The activity of oxidative stress enzymes (catalase, glutathione peroxidase), total antioxidant capacity, as well as the level of heat shock protein were determined. A significant increase in all of the measured parameters was observed after seven weeks of exposure in individuals exposed to higher concentrations of ND (200µgg(-1) food). In animals exposed to lower concentrations of ND (20µgg(-1) food), there were few significant changes to these parameters. Analysis of DNA damage performed after fourteen weeks using the comet assay revealed DNA instabilities in the insects, especially the ones that had been exposed to the higher doses of ND. These findings may suggest that the toxicity of ND is concentration dependent. While high doses interact in a toxic manner, trace amounts, which are more likely in the environment, might be safe for organisms. Extreme caution should be taken when handling nanodiamonds.

Plant stem cells as innovation in cosmetics.

The stem cells thanks to their ability of unlimited division number or transformation into different cell types creating organs, are responsible for regeneration processes. Depending on the organism in which the stem cells exists, they divide to the plant or animal ones. The later group includes the stem cells existing in both embryo's and adult human's organs. It includes, among others, epidermal stem cells, located in the hair follicle relieves and also in its basal layers, and responsible for permanent regeneration of the epidermis. Temporary science looks for method suitable for stimulation of the epidermis stem cells, amongst the other by delivery of e.g., growth factors for proliferation that decrease with the age. One of the methods is the use of the plant cell culture technology, including a number of methods that should ensure growth of plant cells, issues or organs in the environment with the microorganism-free medium. It uses abilities of the different plant cells to dedifferentiation into stem cells and coming back to the pluripotent status. The extracts obtained this way from the plant stem cells are currently used for production of both common or professional care cosmetics. This work describes exactly impact of the plant stem cell extract, coming from one type of the common apple tree (Uttwiler Spätlauber) to human skin as one of the first plant sorts, which are used in cosmetology and esthetic dermatology.

The effect of ingested copper on the structural and cytotoxic properties of Steatoda grossa (Theridiidae) spider silk.

Spiders, assigned to macroconcentrators of heavy metals, are particularly threatened by the toxic effects of these chemicals. Until now, it has not been specified to what extent metals alter the processes proceeding in silk glands and if such changes could consequently influence the chemical and structural properties of the spun web threads. In the present study selected biological properties of Steatoda grossa (Theridiidae) silk yarn after nutritional exposure to copper at sublethal doses (0.234 mM CuSO4) were assessed. It was determined both changes in ultrastructure of ampullate glands and hunting web's architecture as well the cytotoxic effect in model cells (fibroblasts: line ATCC® CCL-1 NCTC clone 929), that were in contact with the analyzed biomaterial. The exposure of spiders to copper caused the occurrence of apoptotic cells in the ampullate glands as well as a significant reduction in the diameter of single fibers in double and multiple connection complexes as compared with control. At both 24 and 72 h of incubation, intensification of apoptotic and necrotic processes was observed in the fibroblast cultures that were remaining in indirect contact with the webs produced by copper-contaminated individuals. In the case of fibroblasts in direct contact with silk from the copper group, a clear cytotoxic effect resulting in an increased frequency of necrosis was observed after 72 h of incubation. The results indicated that copper may change the biological properties of spider silk and compromise its biomaterial properties.

Survival, growth and digestive functions after exposure to nanodiamonds - Transgenerational effects beyond contact time in house cricket strains.

The long-term exposure effects of nanodiamonds (NDs), spanning an organism's entire lifespan and continuing for subsequent generation, remain understudied. Most research has focused on evaluating their biological impacts on cell lines and selected organisms, typically over short exposure durations lasting hours or days. The study aimed to assess growth, mortality, and digestive functions in wild (H) and long-lived (D) strains of Acheta domesticus (Insecta: Orthoptera) after two-generational exposure to NDs in concentrations of 0.2 or 2 mg kg-1 of food, followed by their elimination in the third generation. NDs induced subtle stimulating effect that depended on the strain and generation. In the first generation, more such responses occurred in the H than in the D strain. In the first generation of H strain insects, contact with NDs increased survival, stimulated the growth of young larvae, and the activity of most digestive enzymes in mature adults. The same doses and exposure time did not cause similar effects in the D strain. In the first generation of D strain insects, survival and growth were unaffected by NDs, whereas, in the second generation, significant stimulation of those parameters was visible. Selection towards longevity appears to support higher resistance of the insects to exposure to additional stressor, at least in the first generation. The cessation of ND exposure in the third generation caused potentially harmful changes, which included, e.g., decreased survival probability in H strain insects, slowed growth of both strains, as well as changes in heterochromatin density and distribution in nuclei of the gut cells in both strains. Such a reaction may suggest the involvement of epigenetic inheritance mechanisms, which may become inadequate after the stress factor is removed.

Skin care during the menopause period: noninvasive procedures of beauty studies.

Ageing is a resultant of two processes, including genetically encoded changes in an organism and modifications caused by a negative external environment impact. In the histological aspect, the skin ageing, due to endogenous factors and hormonal changes shows: excessive dryness, Malpighian layer thinning, microcirculation disorders, collagenic or elastin fiber degradation and simultaneous glycation, decreased speed of sebum and perspiration secretion. It is said that skin is a functional picture of the organism and endocrinological system. Any hormone concentration ups and downs may improve its appearance or significantly worsen its condition as well as it may lead to occurrence of dermatological changes. In adult women, the ageing process changes its significance step by step. Despite the passage of time, women want to feel good inside their skins. The adult skin is more requiring and it needs special care, often using a cosmetic apparatus. For better effect and permanent revitalization of the ageing skin, it is recommended to apply properly selected home-use cosmetic preparations. A holistic approach makes it possible to reach the skin density and thickness increase, wrinkles shallowing, humidity and resilience improvement and also recovery of the proper face oval.

Effect of gluten on the digestive tract and fat body of Telodeinopus aoutii (Diplopoda).

Adult specimens or larvae of invertebrates used as food for vertebrates are often maintained close to gluten so they might become vectors for cereal proteins. However, the tissues and internal organs can respond differently in animals with different feeding habits. The midgut epithelium might be a first and sufficient barrier preventing uptake and effects of gluten on the whole body, while the fat body is the main organ that accumulates different xenobiotics. Good models for such research are animals that do not feed on gluten-rich products in their natural environment. The project's goal was to investigate alterations in the midgut epithelium and fat body of the herbivorous millipede Telodeinopus aoutii (Diplopoda) and analyze cell death processes activated by gluten. It enabled us to determine whether changes were intensified or reversed by adaptive mechanisms. Adult specimens were divided into control and experimental animals fed with mushrooms supplemented with gluten and analyzed using transmission electron microscopy, flow cytometry, and confocal microscopy. Two organs were isolated for the qualitative and quantitative analysis: the midgut and the fat body. Our study of the herbivorous T. aoutii which does not naturally feed on gluten containing diet showed that continuous and prolonged gluten feeding activates repair processes that inhibit the processes of cell death (apoptosis and necrosis) and induce an increase in cell viability.

Adaptation by death? Cell death-based tolerance to cadmium in 150-generation exposure of Spodoptera exiqua Hübner (Lepidoptera: Noctuidae).

Mechanisms, including autophagy and apoptosis, which serve to regulate and ensure proper organism functions under optimal conditions, play additional defensive roles under environmental pressure. The aim of this study was to test the following hypotheses: (i) elevated autophagy and apoptosis intensity levels, as defensive processes in response to contact with cadmium, are maintained for a limited number of generations and (ii) the number of generations after which levels of cell death processes reach the reference level depends on selective pressure. Cell death processes were assessed by light and transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL), and cytometric analyses. Model insects (Spodoptera exiqua, Hübner, 1808) were orally exposed to various concentrations of cadmium for 18 generations and compared with reference strains exposed to cadmium or not (control) for over 150 generations. Elevated programmed cell death intensity levels decreased after several generations, indicating tolerance of individuals to cadmium in the diet and verifying the first hypothesis; however, testing the second hypothesis indicated that the number of generations depended not only on pressure intensity, but also on cell death type, since levels of autophagy remained increased for a minimum of 12 generations.

Uncovering nanotoxicity of a water-soluble and red-fluorescent [70]fullerene nanomaterial.

Engineered fullerene materials have attracted the attention of researchers in the biomedical sciences, especially when their synthetic methodology is developed to endow them with significant levels of water-solubility and bioavailability. In this study, we synthesized and characterized a water-soluble and red-fluorescent [70]fullerene nanomaterial, which fluoresced at 693 nm with a quantum yield of 0.065 and a large Stokes shift (around 300 nm). The fullerene nanomaterial generated mainly singlet oxygen after illumination with blue LED light, while superoxide anion radical production was minimal. The transmission electron microscopy as well as fluorescent studies of Drosophila melanogaster revealed that prepared [70]fullerene nanoparticles had better bioavailability than pristine [70]fullerene nanoparticles. The designed nanomaterials were observed in the apical, perinuclear, and basal regions of digestive cells, as well as the basal lamina of the digestive system's epithelium, with no damage to cell organelles and no activation of degenerative processes and cell death. Our findings provide a new perspective for understanding the in vivo behavior of fullerene nanomaterials and their future application in bioimaging and light-activated nanotherapeutics.

Influence of the modifiers in polyol method on magnetically induced hyperthermia and biocompatibility of ultrafine magnetite nanoparticles.

Magnetite nanoparticles (Fe3O4 NPs) are widely tested in various biomedical applications, including magnetically induced hyperthermia. In this study, the influence of the modifiers, i.e., urotropine, polyethylene glycol, and NH4HCO3, on the size, morphology, magnetically induced hyperthermia effect, and biocompatibility were tested for Fe3O4 NPs synthesized by polyol method. The nanoparticles were characterized by a spherical shape and similar size of around 10 nm. At the same time, their surface is functionalized by triethylene glycol or polyethylene glycol, depending on the modifiers. The Fe3O4 NPs synthesized in the presence of urotropine had the highest colloidal stability related to the high positive value of zeta potential (26.03 ± 0.55 mV) but were characterized by the lowest specific absorption rate (SAR) and intrinsic loss power (ILP). The highest potential in the hyperthermia applications have NPs synthesized using NH4HCO3, for which SAR and ILP were equal to 69.6 ± 5.2 W/g and 0.613 ± 0.051 nHm2/kg, respectively. Their application possibility was confirmed for a wide range of magnetic fields and by cytotoxicity tests. The absence of differences in toxicity to dermal fibroblasts between all studied NPs was confirmed. Additionally, no significant changes in the ultrastructure of fibroblast cells were observed apart from the gradual increase in the number of autophagous structures.

Ovaries and testes of Lithobius forficatus (Myriapoda, Chilopoda) react differently to the presence of cadmium in the environment.

Proper reproduction depends on properly functioning gonads (ovaries and testes). Many xenobiotics, including heavy metals, can cause changes in somatic and germ line cells, thus damaging the reproductive capacity. The aim of this study was to investigate the effect of the heavy metal cadmium on the gonads, including germ line and somatic cells. It is important to determine whether cell death processes are triggered in both types of cells in the gonads, and which gonads are more sensitive to the presence of cadmium in the environment. The research was conducted on the soil-dwelling arthropod Lithobius forficatus (Myriapoda, Chilopoda), which is common for European fauna. Animals were cultured in soil supplemented with Cd for different periods (short- and long-term treatment). Gonads were isolated and prepared for qualitative and quantitative analysis, which enabled us to describe all changes which appeared after both the short- and long-term cadmium treatment. The results of our study showed that cadmium affects the structure and ultrastructure of both gonads in soil-dwelling organisms including the activation of cell death processes. However, the male germ line cells are more sensitive to cadmium than female germ line cells. We also observed that germ line cells are protected by the somatic cells of both gonads.

The effect of selected immunostimulants on hemocytes of the false black widow Steatoda grossa (Theridiidae) spiders under chronic exposition to cadmium.

The aim of this study was to analyze whether, and to what extent, long-term exposure to cadmium, administered in sublethal concentrations by the oral route, caused changes in the immune potential of hemocytes in adult female Steatoda grossa spiders. We used artificial and natural immunostimulants, namely phorbol 12-myristate 13-acetate (PMA) and bacterial cell suspension based on Gram-positive (G+, Staphylococcus aureus) and Gram-negative (G-, Pseudomonas fluorescens) bacteria, to compare the status of hemocytes in nonstimulated individuals and those subjected to immunostimulation. After cadmium exposure, the percentage of small nongranular hemocytes in response to G+ cell suspension and PMA mitogen was decreased. Furthermore, in the cadmium-intoxicated spiders the percentage of plasmatocytes after immunostimulation remained lower compared to the complementary control group. Exposure to cadmium also induced several degenerative changes, including typical apoptotic and necrotic changes, in the analyzed types of cells. Immunostimulation by PMA mitogen and G+ bacterial suspension resulted in an increase in the number of cisterns in the rough endoplasmic reticulum of granulocytes, in both the control group and cadmium-treated individuals. These changes were accompanied with a low level of metallothioneins in hemolymph. Chronic cadmium exposure may significantly weaken the immune defense system of spiders during infections.

Hazards related to the presence of cadmium in food - Studies on the European soil centipede, Lithobius forficatus.

The soil is an environment rich in numerous potentially toxic substances/elements when present at elevated concentrations. They can be transported through the successive levels of the trophic chain. Animals living in a contaminated environment or eating contaminated food can accumulate potentially toxic elements in their bodies. One of the potentially toxic metals is cadmium, which accumulates significantly in soils. The aim of our research was to evaluate the changes caused by cadmium supplied with the food administered to invertebrates living in uncontaminated soil. The results were compared with those obtained for animals raised in contaminated soil, where cadmium entered the body via the epidermis. As the material for studies, we chose a common European soil centipede, Lithobius forficatus. Adult specimens were divided into the following experimental groups: C - control animals, Cd12 and Cd45 - animals fed with Chironomus larvae maintained in water containing 80 mg/l CdCl2, for 12 and 45 days, respectively. The material was analyzed using qualitative and quantitative analysis (transmission electron microscopy, confocal microscopy, flow cytometry, atomic absorption spectrometry). Eventually, we can conclude that the digestive system is an effective barrier against the effects of toxic metals on the entire organism, but among the gonads, ovaries are more protected than testes, however, this protection is not sufficient. Accumulation of spherites and mitochondrial alterations are probably involved in survival mechanisms of tissues after Cd intoxication.

Origin of the brushborder in the differentiating midgut of Melasoma saliceti (Chrysomelidae, Coleoptera) embryos.

The embryonic development of Melasoma saliceti takes eight days at room temperature. At the beginning of the 5th day the endoderm cells have already formed a unilayered epithelium of the midgut primordium. The midgut epithelium is formed by flat cells that are not connected by specialized intercellular junctions. Large vesicles can be seen in dilated intercellular spaces of the epithelium. Cytoplasmic projections, similar to microvilli, appear in the vesicles. During the 5th day ofdevelopment, the vesicles grow and become enclosed by the intercellular junctions of a zonula adherens type. During the 6th day of development the cell junctions surrounding the vesicles become transformed into a septate type. On the 8th day of development the vesicles come close to the apical sides of the midgut cells and open towards the yolk. At the same time the microvilli spread over the apical surface of the midgut primordium to form the regular brushborder of the larval midgut. In the species studied the vesicles appear to "prefabricate" the apical surfaces of the future midgut epithelium.

Postembryonic development and differentiation of the midgut in the freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca, Decapoda) larvae.

Neocaridina davidi is a freshwater shrimp that originates from Taiwan and is commonly bred all over the word. Like all decapods, which develop indirectly, this species has pelagic larvae that may differ entirely in their morphology and habits from adult specimens. To fill a gap of knowledge about the developmental biology of freshwater shrimps we decided to document the 3D-localization of the midgut inside the body cavity of larval stages of N. davidi using X-ray microtomography, and to describe all structural and ultrastructural changes of the midgut epithelium (intestine and hepatopancreas) which occur during postembryonic development of N. davidi using light and transmission electron microscopy. We laid emphasis on stem cell functioning and cell death processes connected with differentiation. Our study revealed that while the intestine in both larval stages of N. davidi has the form of a fully developed organ, which resembles that of adult specimens, the hepatopancreas undergoes elongation and differentiation. E-cells, which are midgut stem cells, due to their proliferation and differentiation are responsible for the above-mentioned processes. Our study revealed that apoptosis is a common process in both larval stages of N. davidi in the intestine and proximal region of the hepatopancreas. In zoea III, autophagy as a survival factor is activated in order to protect cells against their death. However, when there are too many autophagic structures in epithelial cells, necrosis as passive cell death is activated. The presence of all types of cell death in the midgut in the zoea III stage confirms that this part of the digestive tract is fully developed and functional. Here, we present the first description of apoptosis, autophagy and necrosis in the digestive system of larval stages of Malacostraca and present the first description of their hepatopancreas elongation and differentiation due to midgut stem cell functioning.

Apoptosis and autophagy in the midgut epithelium of Acheta domesticus (Insecta, Orthoptera, Gryllidae).

The midgut epithelium of Acheta domesticus (Insecta, Orthoptera, Gryllidae), which is composed of columnar digestive cells and regenerative crypts, degenerates in two manners: necrotic and apoptotic. While necrosis was described in our previous paper, programmed cell death was the aim of the present studies. The first morphological signs of programmed cell death in midgut epithelium cells are alterations in the cytoplasm connected with shrinkage of the cells. Gradual modifications in a cell's structure cause it to be discharged into the midgut lumen, where it disintegrates. Autophagy is involved in the disintegration of organelles. The transitions of apoptotic cells are described at the ultrastructural level. Immunostaining methods were used in order to indicate the early stages of apoptosis when DNA fragmentation, which results from apoptotic signaling cascades, occurs.

Fine structure of the midgut epithelium of Nicoletia phytophila Gervais, 1844 (Zygentoma: Nicoletiidae: Nicoletiinae) with special emphasis on its degeneration.

The midgut epithelium of Nicoletia phytophila is composed of columnar digestive cells and regenerative cells that form regenerative nests. The cytoplasm of midgut epithelial cells shows typical regionalization in organelle distribution. Two types of regenerative cells have been distinguished: cells which are able to divide intensively and cells which differentiate. Spot desmosomes have been observed between neighboring regenerative cells. The occurrence of intercellular junctions is discussed. The midgut epithelium degenerates both in an apoptotic and necrotic way. Necrosis proceeds during each molting period (cyclic manner), while apoptosis occurs between each molting, when the midgut epithelium is responsible for e.g. digestion. These processes of epithelium degeneration are described at the ultrastructural level. Our studies not only add new information about fine structure of the midgut epithelium of N. phytophila, but contribute to resolving the relationships within the Zygentoma. There are no doubts about the very close sister position of Nicoletiidae and Ateluridae. The midgut epithelium characters confirm their close relationship. However we do not recommend classifying the atelurid genera only within Nicoletiidae: Nicoletiinae.