Comparing the effects of irradiation with protons or photons on neonatal mouse brain: Apoptosis, oncogenesis and hippocampal alterations.

BACKGROUND AND PURPOSE: Medulloblastoma (MB) is a common primary brain cancer in children. Proton therapy in pediatric MB is intensively studied and widely adopted. Compared to photon, proton radiations offer potential for reduced toxicity due to the characteristic Bragg Peak at the end of their path in tissue. The aim of this study was to compare the effects of irradiation with the same dose of protons or photons in Patched1 heterozygous knockout mice, a murine model predisposed to cancer and non-cancer radiogenic pathologies, including MB and lens opacity. MATERIALS AND METHODS: TOP-IMPLART is a pulsed linear proton accelerator for proton therapy applications. We compared the long-term health effects of 3 Gy of protons or photons in neonatal mice exposed at postnatal day 2, during a peculiarly susceptible developmental phase of the cerebellum, lens, and hippocampus, to genotoxic stress. RESULTS: Experimental testing of the 5 mm Spread-Out Bragg Peak (SOBP) proton beam, through evaluation of apoptotic response, confirmed that both cerebellum and hippocampus were within the SOBP irradiation field. While no differences in MB induction were observed after irradiation with protons or photons, lens opacity examination confirmed sparing of the lens after proton exposure. Marked differences in expression of neurogenesis-related genes and in neuroinflammation, but not in hippocampal neurogenesis, were observed after irradiation of wild-type mice with both radiation types. CONCLUSION: In-vivo experiments with radiosensitive mouse models improve our mechanistic understanding of the dependence of brain damage on radiation quality, thus having important implications in translational research.

Comparative genomics provides insights into molecular adaptation to hypermetamorphosis and cantharidin metabolism in blister beetles (Coleoptera: Meloidae).

Blister beetles (Coleoptera: Meloidae) are currently subdivided into three subfamilies: Eleticinae (a basal group), Nemognathinae, and Meloinae. These are all characterized by the endogenous production of the defensive terpene cantharidin (CA), whereas the two most derived subfamilies show a hypermetamorphic larval development. Here, we provide novel draft genome assemblies of five species sampled across the three blister beetle subfamilies (Iselma pallidipennis, Stenodera caucasica, Zonitis immaculata, Lydus trimaculatus, and Mylabris variabilis) and performed a comparative analysis with other available Meloidae genomes and the closely-related canthariphilous species (Pyrochroa serraticornis) to disclose adaptations at a molecular level. Our results highlighted the expansion and selection of genes potentially responsible for CA production and metabolism, as well as its mobilization and vesicular compartmentalization. Furthermore, we observed adaptive selection patterns and gain of genes devoted to epigenetic regulation, development, and morphogenesis, possibly related to hypermetamorphosis. We hypothesize that most genetic adaptations occurred to support both CA biosynthesis and hypermetamorphosis, two crucial aspects of Meloidae biology that likely contributed to their evolutionary success.

Transition from active motion to anomalous diffusion for Bacillus subtilis confined in hydrogel matrices.

We investigate the motility of B. subtilis under different degrees of confinement induced by transparent porous hydrogels. The dynamical behavior of the bacteria at short times is linked to characteristic parameters describing the hydrogel porosity. Mean squared displacements (MSDs) reveal that the run-and-tumble dynamics of unconfined B. subtilis progressively turns into sub-diffusive motion with increasing confinement. Correspondingly, the median instantaneous velocity of bacteria decreases and becomes more narrowly distributed, while the reorientation rate increases and reaches a plateau value. Analyzing single-trajectories, we show that the average dynamical behavior is the result of complex displacements, in which active, diffusive and sub-diffusive segments coexist. For small and moderate confinements, the number of active segments reduces, while the diffusive and sub-diffusive segments increase. The alternation of sub-diffusion, diffusion and active motion along the same trajectory can be described as a hopping ad trapping motion, in which hopping events correspond to displacements with an instantaneous velocity exceeding the corresponding mean value along a trajectory. Different from previous observations, escape from local trapping occurs for B. subtilis through active runs but also diffusion. Interestingly, the contribution of diffusion is maximum at intermediate confinements. At sufficiently long times transport coefficients estimated from the experimental MSDs under different degrees of confinement can be reproduced using a recently proposed hopping and trapping model. Finally, we propose a quantitative relationship linking the median velocity of confined and unconfined bacteria through the characteristic confinement length of the hydrogel matrix. Our work provides new insights for the bacterial motility in complex media that mimic natural environments and are relevant to important problems like sterilization, water purification, biofilm formation, membrane permeation and bacteria separation.

Toward nano-sized imprinted norepinephrine-derived biopolymer as artificial receptors for detecting IgG1 by surface plasmon resonance.

Bio-based nanostructured molecularly imprinted polymers (nano-MIPs), also known as 'plastibodies', have a real potential to be used as alternatives to natural antibodies. These nanostructures have recently gained significant attention for diagnostic and therapeutic purposes. In this context, we have developed polynorepinephrine (PNE)-based nano-MIPs using an eco-friendly one-pot process for the sensitive and selective detection of a model biomolecule, immunoglobulin IgG1. We first investigated non-imprinted nanostructures (nano-NIPs) based on polydopamine as reference material, using DLS, SEM, and UV-Vis spectroscopy. Subsequently, PNE scaffolds were characterized, both in the form of nano-NIPs and nano-MIPs. Concerning nano-MIPs, we used the epitope-directed imprinting technology to create binding cavities using a small peptide from the constant region of IgG1 as a template. Nano-MIPs were initially immobilized on a sensing surface to assess their binding capacity via surface plasmon resonance (SPR) spectroscopy. This strategy showed very good sensitivity, outperforming planar PNE-based imprinted films while keeping a high selectivity even in complex biological matrices such as human serum. Furthermore, we confirmed the presence of selective binding sites on nano-MIPs by flowing them, along with nano-NIPs, through a microfluidic SPR system, where they interact with the covalently immobilized analyte. This approach resulted in a good imprinting factor of 4.5. Overall, this study underscores the broad potential of these nanostructures as a viable and reusable alternative to antibodies across a variety of bioanalytical, biochemical, and immunohistochemistry analysis techniques.

Nanodiamond Effects on Cancer Cell Radiosensitivity: The Interplay between Their Chemical/Physical Characteristics and the Irradiation Energy.

Nanoparticles are being increasingly studied to enhance radiation effects. Among them, nanodiamonds (NDs) are taken into great consideration due to their low toxicity, inertness, chemical stability, and the possibility of surface functionalization. The objective of this study is to explore the influence of the chemical/physical properties of NDs on cellular radiosensitivity to combined treatments with radiation beams of different energies. DAOY, a human radioresistant medulloblastoma cell line was treated with NDs-differing for surface modifications [hydrogenated (H-NDs) and oxidized (OX-NDs)], size, and concentration-and analysed for (i) ND internalization and intracellular localization, (ii) clonogenic survival after combined treatment with different radiation beam energies and (iii) DNA damage and apoptosis, to explore the nature of ND-radiation biological interactions. Results show that chemical/physical characteristics of NDs are crucial in determining cell toxicity, with hydrogenated NDs (H-NDs) decreasing either cellular viability when administered alone, or cell survival when combined with radiation, depending on ND size and concentration, while OX-NDs do not. Also, irradiation at high energy (γ-rays at 1.25 MeV), in combination with H-NDs, is more efficient in eliciting radiosensitisation when compared to irradiation at lower energy (X-rays at 250 kVp). Finally, the molecular mechanisms of ND radiosensitisation was addressed, demonstrating that cell killing is mediated by the induction of Caspase-3-dependent apoptosis that is independent to DNA damage. Identifying the optimal combination of ND characteristics and radiation energy has the potential to offer a promising therapeutic strategy for tackling radioresistant cancers using H-NDs in conjunction with high-energy radiation.

Sponge-like Cryogels from Liquid-Liquid Phase Separation: Structure, Porosity, and Diffusional Gel Properties.

Macroporous gels find application in several scientific fields, ranging from art restoration to wastewater filtration or cell entrapment. In this work, two-component sponge-like cryogels are challenged to assess their cleaning performances and to investigate how pores size and connectivity affect physico-chemical properties. The gels were obtained through a freeze-thaw process, exploiting a spontaneous polymer-polymer phase-separation occurring in the pre-gel solution. During the freezing step, a highly hydrolyzed polyvinyl alcohol (H-PVA) forms the hydrogel walls. The secondary components, namely a partially hydrolyzed polyvinyl alcohol (L-PVA) or polyvinyl pyrrolidone (PVP), act as modular porogens, being partially extracted during gel washing. H-PVA/L-PVA and H-PVA/PVP mixtures were studied by confocal laser scanning microscopy to unveil sols and gels morphology at the micron-scale, while small angle X-ray scattering was used to get insights about characteristic dimensions at the nanoscale. The gelation mechanism was investigated through rheology measurements, and the characteristic exponents were compared to De Gennes' scaling laws gathered from percolation. In the field of art conservation, these sponge-like gels are ideal systems for the cleaning of artistic painted surfaces. Their interconnected pores allow the diffusion of cleaning fluids at the painted interface, facilitating dirt uptake and/or detachment. This study uncovered a direct relationship linking a gel's cleaning performance to its apparent tortuosity. These findings can pave the way to fine-tuning systems with enhanced cleaning abilities, not restricted to the restoration of irreplaceable priceless works of art, but with possible application in diverse research fields.

Effect of Composition and Freeze-Thaw on the Network Structure, Porosity and Mechanical Properties of Polyvinyl-Alcohol/Chitosan Hydrogels.

We report the synthesis and characterization of poly (vinyl alcohol) (PVA)/Chitosan (CT) cryogels for applications involving the uptake and entrapment of particulate and bacterial colonies. In particular, we systematically investigated the network and pore structures of the gels as a function of CT content and for different freeze-thaw times, combining Small Angle X-Ray Scattering (SAXS), Scanning Electron Microscopy (SEM), and confocal microscopy. The nanoscale analysis obtained from SAXS shows that while the characteristic correlation length of the network is poorly affected by composition and freeze-thaw time, the characteristic size of heterogeneities associated with PVA crystallites decreases with CT content. SEM investigation evidences a transition to a more homogeneous network structure induced by the incorporation of CT that progressively builds a secondary network around the one formed by PVA. A detailed analysis of confocal microscopy image stacks allows to characterize the 3D porosity of the samples, revealing a significantly asymmetric shape of the pores. While the average volume of single pores increases with increasing CT content, the overall porosity remains almost unchanged as a result of the suppression of smaller pores in the PVA network with the progressive incorporation of the more homogeneous CT network. Increasing the freezing time in the FT cycles also results in a decrease of porosity, which can be associated with a growth in the crosslinking of the network due to PVA crystallization. The linear viscoelastic moduli measured by oscillatory rheology show a qualitatively comparable frequency-dependent response in all cases, with a moderate reduction with increasing CT content. This is attributed to changes in the structure of the strands of the PVA network.

Characterization of Early and Late Damage in a Mouse Model of Pelvic Radiation Disease.

Pelvic radiation disease (PRD), a frequent side effect in patients with abdominal/pelvic cancers treated with radiotherapy, remains an unmet medical need. Currently available preclinical models have limited applications for the investigation of PRD pathogenesis and possible therapeutic strategies. In order to select the most effective irradiation protocol for PRD induction in mice, we evaluated the efficacy of three different locally and fractionated X-ray exposures. Using the selected protocol (10 Gy/day × 4 days), we assessed PRD through tissue (number and length of colon crypts) and molecular (expression of genes involved in oxidative stress, cell damage, inflammation, and stem cell markers) analyses at short (3 h or 3 days after X-ray) and long (38 days after X-rays) post-irradiation times. The results show that a primary damage response in term of apoptosis, inflammation, and surrogate markers of oxidative stress was found, thus determining a consequent impairment of cell crypts differentiation and proliferation as well as a local inflammation and a bacterial translocation to mesenteric lymph nodes after several weeks post-irradiation. Changes were also found in microbiota composition, particularly in the relative abundance of dominant phyla, related families, and in alpha diversity indices, as an indication of dysbiotic conditions induced by irradiation. Fecal markers of intestinal inflammation, measured during the experimental timeline, identified lactoferrin, along with elastase, as useful non-invasive tools to monitor disease progression. Thus, our preclinical model may be useful to develop new therapeutic strategies for PRD treatment.

In Vivo Radiobiological Investigations with the TOP-IMPLART Proton Beam on a Medulloblastoma Mouse Model.

Protons are now increasingly used to treat pediatric medulloblastoma (MB) patients. We designed and characterized a setup to deliver proton beams for in vivo radiobiology experiments at a TOP-IMPLART facility, a prototype of a proton-therapy linear accelerator developed at the ENEA Frascati Research Center, with the goal of assessing the feasibility of TOP-IMPLART for small animal proton therapy research. Mice bearing Sonic-Hedgehog (Shh)-dependent MB in the flank were irradiated with protons to test whether irradiation could be restricted to a specific depth in the tumor tissue and to compare apoptosis induced by the same dose of protons or photons. In addition, the brains of neonatal mice at postnatal day 5 (P5), representing a very small target, were irradiated with 6 Gy of protons with two different collimated Spread-Out Bragg Peaks (SOBPs). Apoptosis was visualized by immunohistochemistry for the apoptotic marker caspase-3-activated, and quantified by Western blot. Our findings proved that protons could be delivered to the upper part while sparing the deepest part of MB. In addition, a comparison of the effectiveness of protons and photons revealed a very similar increase in the expression of cleaved caspase-3. Finally, by using a very small target, the brain of P5-neonatal mice, we demonstrated that the proton irradiation field reached the desired depth in brain tissue. Using the TOP-IMPLART accelerator we established setup and procedures for proton irradiation, suitable for translational preclinical studies. This is the first example of in vivo experiments performed with a "full-linac" proton-therapy accelerator.

Orientation of capture antibodies on gold nanoparticles to improve the sensitivity of ELISA-based medical devices.

The sensitivity of ELISA-based devices strongly depends on the right orientation of antibodies on the sensor surface. The aim of this work was to increase the analytical performance of a commercial ELISA-based medical device (VIDAS®), thanks to the specific orientation of antibodies on gold nanostructured disposables. For this purpose, fPSA VIDAS® assay was used as model and the disposable providing the antigen binding surface (SPR®) was functionalized with gold nanostructures coated with monovalent half-fragment antibodies (reduced IgG, rIgG). The functionalization of polystyrene SPRs® with gold nanostructures was achieved through a one-step incubation of gold dispersions in a mixture of non-toxic solvents. Five different concentrations of gold nanoparticles (NPs) were tested with a maximum fluorescence enhancement for NPs density around 3-8 *103 NPs/µm2 (752 ± 11 RFV vs 316 ± 5 RFV of bare SPRs®). The comparison of the dose-response curve obtained with commercial and gold coated-SPRs® revealed a significant improvement (p < 0.0001) of the analytical sensitivity of the VIDAS® system using nanostructured disposables. This improved version of SPRs® allows to distinguish small variations of fPSA concentrations opening the way to the application of this biomarker to other kinds of cancer as recently described in the literature.

Ultrasound and Microwave-Assisted Synthesis of Hexagonally Ordered Ce-Promoted Mesoporous Silica as Ni Supports for Ethanol Steam Reforming.

Solvothermal synthesis of mesoporous materials based on amphiphilic molecules as structure-directing agents can be enhanced using non-conventional technologies for stirring and thermal activation. Here, we disclose a green synthesis approach for the preparation of cerium-modified hexagonally ordered silica sieves. Ultrasound micromixing enabled us to obtain well-dispersed Ce in the self-assembled silica network and yielded ordered materials with high cerium content (Ce/Si molar ratio = 0.08). Microwave dielectric heating, applied by an innovative open-end coaxial antenna, was used to reduce the overall hydrothermal synthesis time and to improve the surface area and textural properties. These mesoporous materials were used as a Ni catalyst support (10 wt.% metal loading) for the ethanol steam reforming reaction. The new catalysts featured complete ethanol conversion, high H2 selectivity (65%) and better stability, compared to the same catalyst prepared with magnetic stirring and conventional heating. The Ce-promoted silica sieves offered a suitable support for the controlled growth of nanocarbon that does not result in catalyst deactivation or poisoning after 6 h on stream.

Conformational changes and location of BSA upon immobilization on zeolitic imidazolate frameworks.

The location and the conformational changes of proteins/enzymes immobilized within Metal Organic Frameworks (MOFs) are still poorly investigated and understood. Bovine serum albumin (BSA), used as a model protein, was immobilized within two different zeolitic imidazolate frameworks (ZIF-zni and ZIF-8). Pristine ZIFs and BSA@ZIFs were characterized by X-ray diffraction, small-angle X-ray scattering, scanning electron microscopy, confocal laser scanning microscopy, thermogravimetric analysis, micro-FTIR and confocal Raman spectroscopy to characterize MOFs structure and the protein location in the materials. Moreover, the secondary structure and conformation changes of BSA after immobilization on both ZIFs were studied with FTIR. BSA is located both in the inner and on the outer surface of MOFs, forming domains that span from the micro- to the nanoscale. BSA crystallinity (ß-sheets + α-helices) increases up to 25 % and 40 % due to immobilization within ZIF-zni and ZIF-8, respectively, with a consequent reduction of ß-turns.

Safety in Rats of a Novel Nasal Spray Formulation for the Prevention of Airborne Viral Infections.

Hexedra+® is a nasal spray containing hydroxypropyl methylcellulose, beta-cyclodextrin, and usnic acid. It has been developed with the aim of reducing the risk of transmission of airborne viral infections, with particular reference to influenza and COVID-19. As part of the preclinical development of the product, we carried out a study on thirty male Wistar rats divided into three study groups and treated with Hexedra+, an alternative formulation containing a double concentration of usnic acid (0.015% instead of 0.0075%) or saline solution. Products were administered at the dose of 30 µL into each nostril, three times a day for seven consecutive days by means of a micropipette. By the end of the treatment period, no significant changes were observed in body weight. Histological examination of nasal mucosa and soft organs did not show any significant difference in the three study groups. Serum transaminase level remained in the normal limit in all the animals treated. The serum level of usnic acid was measured in order to assess the absorption of the molecule through the nasal mucosa. By the end of the study period, the usnic acid serum level was negligible in all the animals treated. In conclusion, the safety profile of Hexedra+ appears favorable in the animal model studied.

Nanostructured bio-based castor oil organogels for the cleaning of artworks.

HYPOTHESIS: Organic solvents are often used for cleaning highly water-sensitive artifacts in modern/contemporary art. Due to the toxicity of most solvents, confining systems must be formulated to use these fluids in a safe and controlled way. We propose here castor oil (CO) organogels, obtained thorough cost-effective sustainable polyurethane crosslinking. This methodology is complementary to previously demonstrated hydrogels, when conservators opt for organic solvents over aqueous formulations. EXPERIMENTS: The gels were characterized via Small-angle Neutron Scattering and rheology before and after swelling in two organic solvents commonly adopted in cleaning paintings. The removal of a photo-aged acrylic-ketonic varnish was evaluated under visible and ultraviolet light, and with FTIR 2D imaging. FINDINGS: The new gels are dry systems that can be easily stored and loaded with solvents before use. Their nanoscale organization, viscoelasticity and cleaning action are controlled changing the amount of crosslinking, the polymeric backbone, and the loaded solvents. The fluids are confined in the nanosized polymeric mesh of the gels, which are highly retentive, granting controlled release over delicate paint layers, and transparent, allowing monitoring of the cleaning process. These features, along with their sustainable synthesis, candidate the CO organogels as feasible solutions for cultural heritage preservation, expanding the palette of advanced tools for conservators over traditional thickeners.

Microgel dynamics within the 3D porous structure of transparent PEG hydrogels.

We report an investigation on the effects of the confinement imposed by application-relevant poly(ethylene glycol) (PEG) hydrogel matrices with controlled porosity on the dynamics of soft microgels. Through a detailed characterization of the internal structure of the hydrogels at the nano and microscale, we were able to link the microgel dynamics, measured by particle tracking, to the 3D geometrical confinement imposed by the porous matrices. PEG hydrogels with a high degree of transparency and tunable pore sizes and volume fractions were obtained using freeze-thawing. We found that the porosity of the hydrogel networks is characterized by elongated channels having asymmetric sections, with the average size decreasing from about 7 to about 2 particle diameters, and the size distribution becoming narrower with increasing PEG content in the pre-reaction mixture. The microgel dynamics slowdown and change from diffusive to sub-diffusive as a result of the increasing confinement. The observed decrease in diffusivity is consistent with models of diffusion in cylindrical pores and can be attributed to hydrodynamic and steric effects in addition to geometrical constriction. A dependence of the effective diffusion coefficient on the pore volume fraction, which is unusually pronounced, suggests the presence of microgel-hydrogel interactions. Our results demonstrate that a detailed characterization of the 3D geometry of the porous network is of primary importance for the understanding of transport properties in complex, random porous media.

2-Butanol Aqueous Solutions: A Combined Molecular Dynamics and Small/Wide-Angle X-ray Scattering Study.

Structural properties of 2-butanol aqueous solutions at different concentrations have been studied using small- and wide-angle X-ray scattering and molecular dynamics simulations. The experimental structure factors have been accurately reproduced by the simulations, allowing one to explain their variation with concentration and to achieve a detailed description of the structural and dynamic properties of the studied systems. The analysis of experimental and computational data has shown that 2-butanol, the simplest aliphatic chiral alcohol, tends to form aggregates at a concentration above 1 M, affecting also both the structural and dynamic properties of the solvent.

Ultra-/Small Angle X-ray Scattering (USAXS/SAXS) and Static Light Scattering (SLS) Modeling as a Tool to Determine Structural Changes and Effect on Growth in S. epidermidis.

Usually, to characterize bacterial cells' susceptibility to antimicrobials, basic microbiology techniques such as serial dilutions or disk assays are used. In this work, we present an approach focused on combining static light scattering (SLS) and ultra-/small angle X-ray scattering (USAXS/SAXS). This approach was used to support microbiology techniques, with the aim of understanding the structural changes caused to bacteria when they are exposed to different stresses like pH, oxidation, and surfactants. Using USAXS/SAXS and SLS data, we developed a detailed multiscale model for a Gram-positive bacterium, S. epidermidis, and we extracted information regarding changes in the overall size and cell thickness induced by different stresses (i.e., pH and hydrogen peroxide). Increasing the concentration of hydrogen peroxide leads to a progressive reduction in cell wall thickness. Moreover, the concomitant use of pH and hydrogen peroxide provides evidence for a synergy in inhibiting the S. epidermidis growth. These promising results will be used as a starting base to further investigate more complex formulations and improve/refine the data modeling of bacteria in the small angle scattering regime.

Molecular Characterization of Kunitz-Type Protease Inhibitors from Blister Beetles (Coleoptera, Meloidae).

Protease inhibitors are widely studied since the unrestricted activity of proteases can cause extensive organ lesions. In particular, elastase activity is involved in the pathophysiology of acute lung injury, for example during SARS-CoV-2 infection, while serine proteases and thrombin-like proteases are involved in the development and/or pathology of the nervous system. Natural protease inhibitors have the advantage to be reversible and with few side effects and thus are increasingly considered as new drugs. Kunitz-type protease inhibitors (KTPIs), reported in the venom of various organisms, such as wasps, spiders, scorpions, and snakes, have been studied for their potent anticoagulant activity and widespread protease inhibitor activity. Putative KTPI anticoagulants have been identified in transcriptomic resources obtained for two blister beetle species, Lydus trimaculatus and Mylabris variabilis. The KTPIs of L. trimaculatus and M. variabilis were characterized by combined transcriptomic and bioinformatics methodologies. The full-length mRNA sequences were divided on the base of the sequence of the active sites of the putative proteins. In silico protein structure analyses of each group of translational products show the biochemical features of the active sites and the potential protease targets. Validation of these genes is the first step for considering these molecules as new drugs for use in medicine.

Lysozyme is Sterically Trapped Within the Silica Cage in Bioinspired Silica-Lysozyme Composites: A Multi-Technique Understanding of Elusive Protein-Material Interactions.

Lysozyme is widely known to promote the formation of condensed silica networks from solutions containing silicic acid, in a reproducible and cost-effective way. However, little is known about the fate of the protein after the formation of the silica particles. Also, the relative arrangement of the different components in the resulting material is a matter of debate. In this study, we investigate the nature of the protein-silica interactions by means of solid-state nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and electron microscopy. We find that lysozyme and silica are in intimate contact and strongly interacting, but their interaction is neither covalent nor electrostatic: lysozyme is mostly trapped inside the silica by steric effects.

Male Accessory Glands of Blister Beetles and Cantharidin Release: A Comparative Ultrastructural Analysis.

Members of the family Meloidae are known to produce cantharidin, a highly toxic monoterpene found in their hemolymph and exuded as droplets capable of deterring many predators. As a nuptial gift, males transfer large amounts of cantharidin to females via a spermatophore, which is formed by specific accessory glands containing high concentrations of this terpene. Using light, electron and ion beam microscopy, the ultrastructural features of the three pairs of male accessory glands as well as the glandular part of the vasa deferentia were comparatively investigated in seven species of blister beetles belonging to five different tribes and two subfamilies. All gland pairs examined share common features such as mesodermal derivation, the presence of muscle sheath, a developed rough endoplasmic reticulum, abundant mitochondria, secretory vesicles, and microvillated apical membranes. Within the same species, glands exhibit distinctive features, suggesting that each pair is responsible for the formation of a specific substance. The vasa deferentia, while showing many similarities within the family, often exhibit features unique to each of the individual species investigated, whereas the accessory glands of the first and second pairs display the highest degree of ultrastructural variability. A comparison across the species shows an interesting constancy limited to ultrastructural features in the third pair of accessory glands. The similarities and differences among the species are discussed in the light of the available literature and in relation to the potential role that blister beetles' male accessory glands could play in the storage and management of cantharidin.