Plasticity of Dental Cell Types in Development, Regeneration, and Evolution.

Recent years have improved our understanding of the plasticity of cell types behind inducing, building, and maintaining different types of teeth. The latest efforts were aided by progress in single-cell transcriptomics, which helped to define not only cell states with mathematical precision but also transitions between them. This includes new aspects of dental epithelial and mesenchymal stem cell niches and beyond. These recent efforts revealed continuous and fluid trajectories connecting cell states during dental development and exposed the natural plasticity of tooth-building progenitors. Such "developmental" plasticity seems to be employed for organizing stem cell niches in adult continuously growing teeth. Furthermore, transitions between mature cell types elicited by trauma might represent a replay of embryonic continuous cell states. Alternatively, they could constitute transitions that evolved de novo, not known from the developmental paradigm. In this review, we discuss and exemplify how dental cell types exhibit plasticity during dynamic processes such as development, self-renewal, repair, and dental replacement. Hypothetically, minor plasticity of cell phenotypes and greater plasticity of transitions between cell subtypes might provide a better response to lifetime challenges, such as damage or dental loss. This plasticity might be additionally harnessed by the evolutionary process during the elaboration of dental cell subtypes in different animal lineages. In turn, the diversification of cell subtypes building teeth brings a diversity of their shape, structural properties, and functions.

Coordinated labio-lingual asymmetries in dental and bone development create a symmetrical acrodont dentition.

Organs throughout the body develop both asymmetrically and symmetrically. Here, we assess how symmetrical teeth in reptiles can be created from asymmetrical tooth germs. Teeth of lepidosaurian reptiles are mostly anchored to the jaw bones by pleurodont ankylosis, where the tooth is held in place on the labial side only. Pleurodont teeth are characterized by significantly asymmetrical development of the labial and lingual sides of the cervical loop, which later leads to uneven deposition of hard tissue. On the other hand, acrodont teeth found in lizards of the Acrodonta clade (i.e. agamas, chameleons) are symmetrically ankylosed to the jaw bone. Here, we have focused on the formation of the symmetrical acrodont dentition of the veiled chameleon (Chamaeleo calyptratus). Intriguingly, our results revealed distinct asymmetries in morphology of the labial and lingual sides of the cervical loop during early developmental stages, both at the gross and ultrastructural level, with specific patterns of cell proliferation and stem cell marker expression. Asymmetrical expression of ST14 was also observed, with a positive domain on the lingual side of the cervical loop overlapping with the SOX2 domain. In contrast, micro-CT analysis of hard tissues revealed that deposition of dentin and enamel was largely symmetrical at the mineralization stage, highlighting the difference between cervical loop morphology during early development and differentiation of odontoblasts throughout later odontogenesis. In conclusion, the early asymmetrical development of the enamel organ seems to be a plesiomorphic character for all squamate reptiles, while symmetrical and precisely orchestrated deposition of hard tissue during tooth formation in acrodont dentitions probably represents a novelty in the Acrodonta clade.

Impact of acute and subchronic inhalation exposure to PbO nanoparticles on mice.

Lead nanoparticles (NPs) are released into air from metal processing, road transport or combustion processes. Inhalation exposure is therefore very likely to occur. However, even though the effects of bulk lead are well known, there is limited knowledge regarding impact of Pb NPs inhalation. This study focused on acute and subchronic exposures to lead oxide nanoparticles (PbO NPs). Mice were exposed to PbO NPs in whole body inhalation chambers for 4-72 h in acute experiment (4.05 × 106 PbO NPs/cm3), and for 1-11 weeks in subchronic experiment (3.83 × 105 particles/cm3 in lower and 1.93 × 106 particles/cm3 in higher exposure group). Presence of NPs was confirmed in all studied organs, including brain, which is very important considering lead neurotoxicity. Lead concentration gradually increased in all tissues depending on the exposure concentration and duration. The most burdened organs were lung and kidney, however liver and brain also showed significant increase of lead concentration during exposure. Histological analysis documented numerous morphological alterations and tissue damage, mainly in lung, but also in liver. Mild pathological changes were observed also in kidney and brain. Levels of glutathione (reduced and oxidized) were modulated mainly in lung in both, acute and subchronic exposures. Increase of lipid peroxidation was observed in kidney after acute exposure. This study characterized impacts of short to longer-term inhalation exposure, proved transport of PbO NPs to secondary organs, documented time and concentration dependent gradual increase of Pb concentration and histopathological damage in tissues.

Ciliopathy Protein Tmem107 Plays Multiple Roles in Craniofacial Development.

A broad spectrum of human diseases called ciliopathies is caused by defective primary cilia morphology or signal transduction. The primary cilium is a solitary organelle that responds to mechanical and chemical stimuli from extracellular and intracellular environments. Transmembrane protein 107 (TMEM107) is localized in the primary cilium and is enriched at the transition zone where it acts to regulate protein content of the cilium. Mutations in TMEM107 were previously connected with oral-facial-digital syndrome, Meckel-Gruber syndrome, and Joubert syndrome exhibiting a range of ciliopathic defects. Here, we analyze a role of Tmem107 in craniofacial development with special focus on palate formation, using mouse embryos with a complete knockout of Tmem107. Tmem107-/- mice were affected by a broad spectrum of craniofacial defects, including shorter snout, expansion of the facial midline, cleft lip, extensive exencephaly, and microphthalmia or anophthalmia. External abnormalities were accompanied by defects in skeletal structures, including ossification delay in several membranous bones and enlargement of the nasal septum or defects in vomeronasal cartilage. Alteration in palatal shelves growth resulted in clefting of the secondary palate. Palatal defects were caused by increased mesenchymal proliferation leading to early overgrowth of palatal shelves followed by defects in their horizontalization. Moreover, the expression of epithelial stemness marker SOX2 was altered in the palatal shelves of Tmem107-/- animals, and differences in mesenchymal SOX9 expression demonstrated the enhancement of neural crest migration. Detailed analysis of primary cilia revealed region-specific changes in ciliary morphology accompanied by alteration of acetylated tubulin and IFT88 expression. Moreover, Shh and Gli1 expression was increased in Tmem107-/- animals as shown by in situ hybridization. Thus, TMEM107 is essential for proper head development, and defective TMEM107 function leads to ciliary morphology disruptions in a region-specific manner, which may explain the complex mutant phenotype.

Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs.

BACKGROUND: Lead is well known environmental pollutant, which can cause toxic effects in multiple organ systems. However, the influence of lead oxide nanoparticles, frequently emitted to the environment by high temperature technological processes, is still concealed. Therefore, we investigate lead oxide nanoparticle distribution through the body upon their entry into lungs and determine the microscopic and ultramicroscopic changes caused by the nanoparticles in primary and secondary target organs. METHODS: Adult female mice (ICR strain) were continuously exposed to lead oxide nanoparticles (PbO-NPs) with an average concentration approximately 106 particles/cm3 for 6 weeks (24 h/day, 7 days/week). At the end of the exposure period, lung, brain, liver, kidney, spleen, and blood were collected for chemical, histological, immunohistochemical and electron microscopic analyses. RESULTS: Lead content was found to be the highest in the kidney and lungs, followed by the liver and spleen; the smallest content of lead was found in brain. Nanoparticles were located in all analysed tissues and their highest number was found in the lung and liver. Kidney, spleen and brain contained lower number of nanoparticles, being about the same in all three organs. Lungs of animals exposed to lead oxide nanoparticles exhibited hyperaemia, small areas of atelectasis, alveolar emphysema, focal acute catarrhal bronchiolitis and also haemostasis with presence of siderophages in some animals. Nanoparticles were located in phagosomes or formed clusters within cytoplasmic vesicles. In the liver, lead oxide nanoparticle exposure caused hepatic remodeling with enlargement and hydropic degeneration of hepatocytes, centrilobular hypertrophy of hepatocytes with karyomegaly, areas of hepatic necrosis, occasional periportal inflammation, and extensive accumulation of lipid droplets. Nanoparticles were accumulated within mitochondria and peroxisomes forming aggregates enveloped by an electron-dense mitochondrial matrix. Only in some kidney samples, we observed areas of inflammatory infiltrates around renal corpuscles, tubules or vessels in the cortex. Lead oxide nanoparticles were dispersed in the cytoplasm, but not within cell organelles. There were no significant morphological changes in the spleen as a secondary target organ. Thus, pathological changes correlated with the amount of nanoparticles found in cells rather than with the concentration of lead in a given organ. CONCLUSIONS: Sub-chronic exposure to lead oxide nanoparticles has profound negative effects at both cellular and tissue levels. Notably, the fate and arrangement of lead oxide nanoparticles were dependent on the type of organs.

Role of Primary Cilia in Odontogenesis.

Primary cilium is a solitary organelle that emanates from the surface of most postmitotic mammalian cells and serves as a sensory organelle, transmitting the mechanical and chemical cues to the cell. Primary cilia are key coordinators of various signaling pathways during development and maintenance of tissue homeostasis. The emerging evidence implicates primary cilia function in tooth development. Primary cilia are located in the dental epithelium and mesenchyme at early stages of tooth development and later during cell differentiation and production of hard tissues. The cilia are present when interactions between both the epithelium and mesenchyme are required for normal morphogenesis. As the primary cilium coordinates several signaling pathways essential for odontogenesis, ciliary defects can interrupt the latter process. Genetic or experimental alterations of cilia function lead to various developmental defects, including supernumerary or missing teeth, enamel and dentin hypoplasia, or teeth crowding. Moreover, dental phenotypes are observed in ciliopathies, including Bardet-Biedl syndrome, Ellis-van Creveld syndrome, Weyers acrofacial dysostosis, cranioectodermal dysplasia, and oral-facial-digital syndrome, altogether demonstrating that primary cilia play a critical role in regulation of both the early odontogenesis and later differentiation of hard tissue-producing cells. Here, we summarize the current evidence for the localization of primary cilia in dental tissues and the impact of disrupted cilia signaling on tooth development in ciliopathies.

Statins do not inhibit the FGFR signaling in chondrocytes.

OBJECTIVE: Statins are widely used drugs for cholesterol lowering, which were recently found to counteract the effects of aberrant fibroblast growth factor receptor (FGFR3) signaling in cell and animal models of FGFR3-related chondrodysplasia. This opened an intriguing therapeutic possibility for human dwarfing conditions caused by gain-of-function mutations in FGFR3, although the mechanism of statin action on FGFR3 remains unclear. Here, we determine the effect of statins on FGFR signaling in chondrocytes. DESIGN: Cultured chondrocyte cell lines, mouse embryonic tibia cultures and limb bud micromasses were treated with FGF2 to activate FGFR signaling. The effects of atorvastatin, fluvastatin, lovastatin and pravastatin on FGFR3 protein stability and on FGFR-mediated chondrocyte growth-arrest, loss of extracellular matrix (ECM), induction of premature senescence and hypertrophic differentiation were evaluated. RESULTS: Statins did not alter the level of FGFR3 protein expression nor produce any effect on FGFR-mediated inhibition of chondrocyte proliferation and hypertrophic differentiation in cultured chondrocyte cell lines, mouse tibia cultures or limb bud micromasses. CONCLUSION: We conclude that statins do not inhibit the FGFR signaling in chondrocytes. Therefore the statin-mediated rescue of FGFR3-related chondrodysplasia, described before, is likely not intrinsic to the growth plate cartilage.

Osteogenic Potential of the Transcription Factor c-MYB.

The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

Impact of acute and chronic inhalation exposure to CdO nanoparticles on mice.

Cadmium nanoparticles can represent a risk in both industrial and environmental settings, but there is little knowledge on the impacts of their inhalation, especially concerning longer-term exposures. In this study, mice were exposed to cadmium oxide (CdO) nanoparticles in whole body inhalation chambers for 4 to 72 h in acute and 1 to 13 weeks (24 h/day, 7 days/week) in chronic exposure to investigate the dynamics of nanoparticle uptake and effects. In the acute experiment, mice were exposed to 2.95 × 106 particles/cm3 (31.7 µg CdO/m3). The same concentration and a lower one (1.18 × 106 particles/cm3, 12.7 µg CdO/m3) were used for the chronic exposure. Transmission electron microscopy documented distribution of nanoparticles into all studied organs. Major portion of nanoparticles was retained in the lung, but longer exposure led to a greater relative redistribution into secondary organs, namely the kidney, and also the liver and spleen. Accumulation of Cd in the lung and liver occurred already after 24 h and in the brain, kidney, and spleen after 72 h of exposure, and a further increase of Cd levels was observed throughout the chronic exposure. There were significant differences in both Cd accumulation and effects between the two exposure doses. Lung weight in the higher exposure group increased up to 2-fold compared to the control. Histological analyses showed dose-dependent alterations in lung and liver morphology and damage to their tissue. Modulation of oxidative stress parameters including glutathione levels and increased lipid peroxidation occurred mainly after the greater chronic exposure. The results emphasize risk of longer-term inhalation of cadmium nanoparticles, since adverse effects occurring after shorter exposures gradually progressed with a longer exposure duration.

Role of c-Myb in chondrogenesis.

The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation.

Properties of neural crest-like cells differentiated from human embryonic stem cells.

Neural crest cells (NCCs) derive early in vertebrate ontogenesis from neural tube as a population of migratory cells with exquisite differentiation potential. Abnormalities in NCC behaviour are cause of debilitating diseases including cancers and a spectrum of neurocristopathies. Thanks to their multilineage differentiation capacity NCCs offer a cell source for regenerative medicine. Both these aspects make NCC biology an important issue to study, which can currently be addressed using methodologies based on pluripotent stem cells. Here we contributed to understanding the biology of human NCCs by refining the protocol for differentiation/propagation of NCClike cells from human embryonic stem cells and by characterizing the molecular and functional phenotype of such cells. Most importantly, we improved formulation of media for NCC culture, we found that poly-L-ornithine combined with fibronectin provide good support for NCC growth, we unravelled the tendency of cultured NCCs to maintain heterogeneity of CD271 expression, and we showed that NCCs derived here possess the capacity to react to BMP4 signals by dramatically up-regulating MSX1, which is linked to odontogenesis.

Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles.

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.

Radiography, computed tomography and magnetic resonance imaging of craniofacial structures in pig.

The pig has recently become popular as a large animal experimental model in many fields of biomedical research. The aim of this study is to evaluate the basic anatomical structures in the head region of the pig to lay the groundwork for its practical clinical usage or pre-clinical research in the future. We used three different diagnostic imaging methods: radiography, computed tomography (CT) and magnetic resonance imaging (MRI). The analysis showed that radiographic imaging is suitable only for general evaluation of the facial area of the pig skull. CT images showed excellent spatial definition of bony structures of the whole craniofacial area, and MRI images revealed fine soft tissue details. Radiography is preferentially suited to general assessment of bone structures of the facial skeleton; however, the thick layer of adipose tissue in the craniofacial region of the pig makes the imaging of some parts difficult or even impossible. CT is useful for revealing morphological details of mineralized tissues, whereas MRI is more suitable for soft tissue analysis and the detection of subtle pathologic changes in both bone and soft tissues. Therefore, before using pigs as an experimental model in craniofacial research, it is necessary to evaluate the suitability and disadvantages of potential imaging methods and how appropriate they are for accurate visualization of desired structures.

Expression, function and regulation of Evi-1 during embryonic avian development.

Ecotropical viral integration site 1 (Evi-1) is a transcription factor essential for vascularisation and cell proliferation during embryonic development. The chimeric transcription factor AML1-EVI-1 is activated in leukaemia where it plays a role as a differentiation block and stimulator of proliferation. Here, we cloned chicken Evi-1 and analysed its expression during embryonic development. There was early expression in the pharyngeal arches, in the brain and intermediate mesoderm of chicken embryos at stage 15. Later at stage 20, Evi-1 mesenchymal expression was concentrated in the second pharyngeal arch, and weaker expression was found in the mandibular and maxillary prominences. Facial expression decreased in intensity during development. Evi-1 expression in the limb was also limited to the mesenchyme with the most prominent expression in the anterior margin. Evi-1 was not detectable in the posterior limb bud. At later stages, Evi-1 was expressed in the peripheral mesenchyme of the limb but not in the developing precartilage blastema. At stage 29, the expression became restricted to the perichondrium and interdigital areas; however, the cartilage condensations themselves were negative. To study the function of Evi-1 in chondrogenesis, we knocked down expression in limb micromass cultures using siRNA. Chondrogenesis was significantly reduced in both anterior and posterior cultures. Since Evi-1 was expressed adjacent to the apical ectodermal ridge and this area is a source of FGFs, we tested whether endogenous FGF receptor signalling was necessary to maintain its expression. Inhibitors of FGFRs (PD161570 and SU5402) were applied to wing mesenchyme, and downregulation of Evi-1 expression was observed after treatment with both inhibitors. Therefore, Evi-1 may be a transcription factor mediating the effects of FGF and may also be defining the size of cartilage elements in the limb.

Investigation of an autologous blood treatment strategy for temporomandibular joint hypermobility in a pig model.

Many different surgical and non-surgical techniques are used for the treatment of temporomandibular joint (TMJ) hypermobility. One of these methods is autologous blood injection into the TMJ. The fate of the autologous blood used for treatment of recurring condylar dislocation is still not completely understood. The authors used 12 pigs (Sus scrota f. domestica) as a model species for autologous blood delivery into the TMJ. Blood injection was followed by histopathological analysis at different times after treatment (1h, 1, 2 and 4 weeks). Samples were examined by magnetic resonance imaging, macroscopic and histological methods. The deposition of the remaining blood was observed in the form of clots in the distal parts of the upper joint cavity 1h and 1 week after treatment. 2 weeks after treatment, small blood clots were still apparent in the distal part of the upper joint cavity. 4 weeks after surgery, no remnants of blood, changes or adhesions were apparent inside the TMJ. No morphological or histological changes were observed in the TMJ after the injection of autologous blood suggesting another mechanism is involved in the hypermobility treatment.

The pig as an experimental model for clinical craniofacial research.

The pig represents a useful, large experimental model for biomedical research. Recently, it has been used in different areas of biomedical research. The aim of this study was to review the basic anatomical structures of the head region in the pig in relation to their use in current research. Attention was focused on the areas that are frequently affected by pathological processes in humans: the oral cavity with teeth, salivary gland, orbit, nasal cavity and paranasal sinuses, maxilla, mandible and temporomandibular joint. Not all of the structures have an equal morphology in the pig and human, and these morphological dissimilarities must be taken into account before choosing the pig as an experimental model for regenerative medicine.

Early regression of the dental lamina underlies the development of diphyodont dentitions.

Functional tooth germs in mammals, reptiles, and chondrichthyans are initiated from a dental lamina. The longevity of the lamina plays a role in governing the number of tooth generations. Monophyodont species have no replacement dental lamina, while polyphyodont species have a permanent continuous lamina. In diphyodont species, the dental lamina fragments and regresses after initiation of the second tooth generation. Regression of the lamina seems to be an important mechanism in preventing the further development of replacement teeth. Defects in the complete removal of the lamina lead to cyst formation and has been linked to ameloblastomas. Here, we show the previously unknown mechanisms behind the disappearance of the dental lamina, involving a combination of cell migration, cell-fate transformation, and apoptosis. Lamina regression starts with the loss of the basement membrane, allowing the epithelial cells to break away from the lamina and migrate into the surrounding mesenchyme. Cells deactivate epithelial markers (E-cadherin, cytokeratin), up-regulate Slug and MMP2, and activate mesenchymal markers (vimentin), while residual lamina cells are removed by apoptosis. The uncovering of the processes behind lamina degradation allows us to clarify the evolution of diphyodonty, and provides a mechanism for future manipulation of the number of tooth generations.

Gene discovery in craniofacial development and disease--cashing in your chips.

An unbiased, polygenic approach is needed to unravel the complex molecular bases of craniofacial development and disease. DNA microarrays, the current paradigm of genome-wide analysis, permit the simultaneous study of many thousands of genes, the ready identification of candidate molecules and pathways, and the compilation of gene expression profiles for whole systems--pathologic and embryonic alike. We survey the existing literature applying microarrays to craniofacial biology and highlight the value of animal models, particularly mice and chickens, to understanding molecular regulation in the craniofacial complex. We also emphasize the importance of functional studies and high-throughput assays to extracting useful data from microarray output. It is our goal to help put researchers and clinicians on the same page as microarray technology moves into the forefront of craniofacial biology.

[Cantharidin and its detection in forensic toxicology]. / Cantharidin a jeho prukaz ve forenzní toxikologii.

Experimental detection of deadly toxic cantharidin C10H12O4 by chemical analysis was performed in imagoes of two species of cryptotoxic Meloidae beetles--Lytta vesicatoria and Mylabris variabilis. Samples were processed by extraction and sublimation and analyzed by gas chromatography. Graphic documentation proved the presence of cantharidin. A part of microcristalline sublimate was studied microscopically and determinant features of crystalline cantharidin were identified. Moreover, cantharidin levels in single body parts of the beetle were investigated.