Laser-driven x-ray and proton micro-source and application to simultaneous single-shot bi-modal radiographic imaging.

Radiographic imaging with x-rays and protons is an omnipresent tool in basic research and applications in industry, material science and medical diagnostics. The information contained in both modalities can often be valuable in principle, but difficult to access simultaneously. Laser-driven solid-density plasma-sources deliver both kinds of radiation, but mostly single modalities have been explored for applications. Their potential for bi-modal radiographic imaging has never been fully realized, due to problems in generating appropriate sources and separating image modalities. Here, we report on the generation of proton and x-ray micro-sources in laser-plasma interactions of the focused Texas Petawatt laser with solid-density, micrometer-sized tungsten needles. We apply them for bi-modal radiographic imaging of biological and technological objects in a single laser shot. Thereby, advantages of laser-driven sources could be enriched beyond their small footprint by embracing their additional unique properties, including the spectral bandwidth, small source size and multi-mode emission.

Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane.

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200 µm, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6 days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications.

Embryological evidence substantiates the subcoxal theory on the origin of pleuron in insects.

The lateral body plate pleuron is a significant structure in insects that contributes to the development and elaboration of wings and limbs (appendages). Although the pleuron is thought to originate from the proximal-most appendicular segment, the subcoxa, details remain unclear, and the morphological boundary between the dorsal body plate tergum and appendage (BTA) has not been clearly specified. Employing low-vacuum scanning electron microscopy (SEM) and the nano-suit method for SEM, we followed, in detail, the development of the thoracic segments of the two-spotted cricket Gryllus bimaculatus and succeeded in clearly defining the BTA. This study demonstrates the subcoxal origin of the pleuron, suggests the tergal origin of spiracles, and reveals that the wing proper originates exclusively from the tergum, whereas the wing hinge and direct muscles may be appendicular in origin, suggesting the dual origin (i.e., tergal plus appendicular origin) of wings.

Genital Autocleaning in the Male Cricket Gryllus bimaculatus (2): Rhythmic Movements of the Genitalia and Their Motor Control.

Three types of genital movement, their neural controls, and functional roles were investigated to gain a better understanding of the mechanism underlying autocleaning in the male cricket. The membrane complex consisting of the median pouch and genital chamber floor shows peculiar undulation that is composed of two types of movements: a right-left large shift and small crease-like movements. The large shift was caused by contraction of a pair of muscles (MPA) located anterior to the median pouch, while the crease-like movements were caused by numerous muscle fibers extending over the membrane complex. The MPA and muscle fibers were each innervated by efferent neurons in the terminal abdominal ganglion. Experiments with artificial dirt mimicking a foreign object revealed that the crease-like movements were responsible for dirt transport, while the large shift participated in sweeping the dirt into the lateral pouch as a trash container. On the other hand, the dorsal pouch serving as a template for the spermatophore showed a jerky bending movement. Simultaneous monitoring of the membrane complex and dorsal pouch activities suggested that their movements cooperate to enable the efficient evacuation of waste in the dorsal pouch. Based on the results, we conclude that genital autocleaning supports the production of the spermatophore.

Genital Autocleaning in the Male Cricket Gryllus bimaculatus (1): Structure and Function of the Genital Membrane.

We found that the genitalia of the male cricket Gryllus bimaculatus are equipped with an autocleaning system. The cricket keeps its genitalia clean by removing foreign matter and endogenous waste. Morphological study showed that the membrane complex consists of a median pouch and a genital chamber floor covered by small scales, each of which has a base of approximately 10 µm in width and a fringe with 5-10 spines 3-20 µm in length. The scales are arranged symmetrically about the midline, curving gradually in the lateral direction and continuing to the lateral pouch serving as a trash container. Observation of cleaning revealed that a small piece of artificial dirt placed on the membrane complex was conveyed over a distance of 1.3 mm to the lateral pouch in 12 minutes. Inspection of the dorsal pouch just after spermatophore extrusion in the mating stage revealed that there were patchy remnants of spermatophore material on the inner surface of the pouch, but that these were evacuated in a few minutes. Surgical elimination of the median pouch caused the formation of abnormal spermatophores with the sperm tube and attachment plate being deformed. These results suggest that genital autocleaning is indispensable for the production of a normal spermatophore in the male cricket.

Morphology and development of the accessory glands in various female cricket species.

The study presents new results with regard to the morphometric and ultrastructural development of the accessory glands in females of the three cricket species Gryllus bimaculatus, Gryllus assimilis, and Acheta domesticus. Furthermore, possible age-dependence of secretory productivity of single organs was analyzed by application of the ligature technique introduced in a previous contribution. Within the first 12 days of the adult phase, the accessory glands of all investigated cricket species exhibit a significant increase in length and width which assumes values between 50 and 100%. This gland growth is rather the result of a continuous increase in cellular volume and less that of mitotic cell propagation. In all species height and width of single gland cells increase by 60-80% within the studied time interval. These changes in morphometry are commonly accompanied by ultrastructural modifications. Total glandular secretion is subject to an increase from the 5th to the 12th day of adult age. This development corresponds well with the number of eggs contemporaneously oviposited into the substrate and thus underlines the hypothesis, according to which the main function of the secretion consists in acting as a lubricant for the facilitated transport of the oocytes through the ovipositor.

Fine structure of synaptic sites and circuits in mushroom bodies of insect brains.

In the insect brain, mushroom bodies represent a prominent central neuropil for multisensory integration and, crucially, for learning and memory. For this reason, special attention has been focused on its small chemical synapses. Early studies on synaptic types and their distribution, using conventional electron microscopy, and recent publications have resolved basic features of synaptic circuits. More recent studies, using experimental methods for resolving neurons, such as immunocytochemistry, genetic labelling, high resolution confocal microscopy and more advanced electron microscopy, have revealed many new details about the fine structure and molecular contents of identifiable neurons of mushroom bodies and has led to more refined modelling of functional organisation. Synaptic circuitries have been described in most detail for the calyces. In contrast, the mushroom bodies' columnar peduncle and lobes have been explored to a lesser degree. In dissecting local microcircuits, the scientist is confronted with complex neuronal compartmentalisation and specific synaptic arrangements. This article reviews classical and modern studies on the fine structure of synapses and their networks in mushroom bodies across several insect species.

Micromorphological differentiation of left and right stridulatory apparatus in crickets (Orthoptera: Gryllidae).

The present study compared micromorphological differentiation of stridulatory apparatus between the functional right tegmen and non-functional left tegmen, analyzed under scanning electron microscope (SEM), among eight Gryllidae species. The results showed that the main differences were found on the length and shape of files and teeth. The length of stridulatory file and teeth number were lower on the left stridulatory apparatus than that on right stridulatory apparatus in all species. However, the ratio between the length of stridulatory teeth and the interval of stridulatory teeth was significantly higher on the left stridulatory apparatus than that on right stridulatory apparatus in Dianemobius fascipes, Polionemobius taprobanensis, Pteronemobius gifuensis, Teleogryllus occipitalis and Oecanthus longicauda. In addition, the length of stridulatory teeth was positively related to number of stridulatory teeth, however, the interval of stridulatory teeth was negatively related to the ratio between the length of stridulatory teeth and the interval of stridulatory teeth for left and right stridulatory apparatus. Our result illustrated that the length of left and right stridulatory file and teeth length could be an effective character to distinguish species. Left stridulatory apparatus was not entirely degraded than right stridulatory apparatus.

Mouthparts and nectar feeding of the flower visiting cricket Glomeremus orchidophilus (Gryllacrididae).

Glomeremus orchidophilus (Gryllacrididae) is a flower visiting cricket on the tropical island La Réunion. This species is the only Orthoptera shown to be a pollinator of a plant. We studied its nectar feeding behavior and mouthpart morphology in detail. Since G. orchidophilus possesses biting-and-chewing mouthparts, our objective was to find behavioral and/or structural specializations for nectar-feeding. The comparative analysis of feeding behavior revealed that fluid is taken up without movements of the mouthparts in Glomeremus. A comparative morphological examination of two Glomeremus species, together with several representatives of other Gryllacrididae and other Ensifera taxa revealed subtle adaptations to fluid feeding in Glomeremus. All representatives of Gryllacrididae were found to possess a distinct patch of microtrichia at the tip of their galeae. However, in Glomeremus a channel is formed between the distal components of the maxillae and the mandibles on each side of the body. Micro-CT and SEM examination revealed a longitudinal groove that extends over the galea beginning at the patch of microtrichia in the studied Glomeremus species. We hypothesize that the microtrichia take up fluid by capillarity and the action of the cibarium and pharyngeal pumps transports fluid along the channels between the maxillae and mandibles into the preoral cavity. These mouthpart features allow nectar uptake from flowers that is unique in Orthoptera.

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.

Ultrastructure of the gut epithelium in Acheta domesticus after long-term exposure to nanodiamonds supplied with food.

The biosafety of nanoparticles and the potential toxicity of nanopollutants and/or nanowastes are all currently burning issues. The increased use of nanoparticles, including nanodiamonds (ND), entails the real risk of their penetration into food chains, which may result in the contamination of animal and, as a result, human food. Knowledge about changes in the ultrastructure of tissues in organisms that have been exposed to ND is still very limited. The aim of the study was to describe the ultrastructure of the gut epithelium in Acheta domesticus after exposure to different concentrations of ND (0, 20 or 200 µg g(-1) - control, ND20 and ND200 groups, respectively) administered with food over a five-week period. The ultrastructure of the foregut, midgut and hindgut was assessed using Transmission Electron Microscopy (TEM). A number of changes in the structure of the gut in crickets that had consumed nanodiamond-contaminated food were observed. The epithelium of the midgut and hindgut were clearly damaged by ND, although the foregut was not affected. A positive relationship between the ND concentration in food and the degree of damage to the structure of epithelial cells was observed. Autophagy, especially mitophagy and reticulophagy, was activated in relation to the appearance of ND particles. A putative ND toxicity mechanizm is proposed. Extreme caution should be maintained when using nanodiamonds on a large scale.

Autophagy in the fat body cells of the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae, Saltatoria) during overwintering.

The cave cricket Troglophilus neglectus regularly overwinters for 4-5 months in hypogean habitats. Winter dormancy is a natural starvation period, providing the opportunity to study autophagy under natural conditions. We aimed to evaluate the autophagic activity in adipocytes and urocytes of the fat body in three time frames: directly before overwintering, in the middle of dormancy, and at its end. For this purpose, we sampled individuals in caves. The cell ultrastructure was studied by transmission electron microscopy (TEM) and the abundance of autophagosomes by immunofluorescence microscopy (IFM), applying the widely used, specific immunolabeling marker microtubule-associated protein 1 light chain 3 (LC3). Before overwintering, TEM revealed scarce autophagosomes and residual bodies in the adipocytes and none in the urocytes. Congruently, IFM showed a very limited or no reaction. In the middle and at the end of overwintering, in both cell types, phagophores, autophagosomes, autolysosomes, and residual bodies were identified by TEM, while LC3 immunolabeling for detecting autophagosomes showed a conspicuous positive reaction. Both methods revealed that there were no significant differences between the sexes in any time frame. Minimal autophagic activity was detected before the winter dormancy, and it gradually intensified till the end of overwintering, probably because reserve proteins in protein granula are not composed of all the required amino acids. We conclude that in T. neglectus, autophagy is a substantial response to starvation and supports homeostatic processes during winter dormancy by supplying cells with nutrients.

Light and electron microscopy study of the spermatheca of Eupholidoptera chabrieri bimucronata (Ramme, 1927) and Uromenus brevicollis trinacriae La Greca 1964 (Orthoptera: Tettigoniidae).

A study by both optical and electron microscopy has been carried out on the spermatheca of Eupholidoptera chabrieri bimucronata and Uromenus brevicollis trinacriae (Orthoptera: Tettigoniidae). In both the examined species, the spermatheca consists of a sac/kidney-shaped seminal receptacle and a more or less tortuous spermathecal duct that opens into the common oviduct. The wall of both the organs consists of a pseudostratified epithelium surmounted by a cuticular intima; the latter is made up of a thicker endocuticle and an epicuticle. The epithelium shows two different cell types, irregularly arranged and with well differentiated functions: cuticle-forming and gland cells. In both the species, the cuticle-forming cells perform other functions, in addition to producing the cuticular intima. The gland cells never come in contact with the cuticular intima, have inside the reservoir a secretion whose appearance can diversify also in contiguous zones of the seminal receptacle. Based on our findings in both the species, the functions of the seminal receptacle would differ from those of the spermathecal duct. In the latter, some areas of the wall of the connecting tract show an activity of lysis, by contiguous epithelial cells, that could play a role in control and selection of spermatozoa. As for the feather-shaped spermatodesms, similar in both the species, freeze-fracture observations have shown that the acrosome of each spermatozoon regularly covers three-quarters of the extension of the acrosome of the following spermatozoon. Finally, the significance of our findings, compared with what is known in literature, is discussed.

Nanomechanical properties of wing membrane layers in the house cricket (Acheta domesticus Linnaeus).

Many insect wings change shape dynamically during the wingbeat cycle, and these deformations have the potential to confer energetic and aerodynamic benefits during flight. Due to the lack of musculature within the wing itself, the changing form of the wing is determined primarily by its passive response to inertial and aerodynamic forces. This response is in part controlled by the wing's mechanical properties, which vary across the membrane to produce regions of differing stiffness. Previous studies of wing mechanical properties have largely focused on surface or bulk measurements, but this ignores the layered nature of the wing. In our work, we investigated the mechanical properties of the wings of the house cricket (Acheta domesticus) with the aim of determining differences between layers within the wing. Nanoindentation was performed on both the surface and the interior layers of cross-sectioned samples of the wing to measure the Young's modulus and hardness of the outer- and innermost layers. The results demonstrate that the interior of the wing is stiffer than the surface, and both properties vary across the wing.

Morphology and ultrastructure of the accessory glands in the female genital tract of the house cricket, Acheta domesticus.

The accessory glands in the genital tract of female Acheta domesticus L. (Orthoptera: Gryllidae) were investigated in detail. The glands are situated within the 7(th) and 8(th) abdominal segment and lead to the genital chamber lateral to the terminal papilla of the ductus receptaculi. The shape of the gland is characterized by a complex system of tubules, including numerous ramifications. The gland's size ranges from 2 to 4 mm. The epithelium is constructed according to a simple scheme and consists of a cuticular intima at the luminal side, one layer of gland cells, and a basallamina at the outermost side. The observed morphology of the accessory glands widely corresponds with that in other cricket species (e.g., Teleogryllus commodus). This is also true for the structure of a single gland cell, which can be subdivided into a basal part with nucleus and intracellular cisternae, as well as an apical part with all those compartments responsible for the production of the secretion. The secretion itself may be classified as lipophilic and is produced for the first time 4 to 6 days after the imaginai moult. Several endogenic functions of the secretion are discussed (lubricant for oviposition, support for introducing the tube of the spermatophore into the ductus receptaculi, etc.).

The morphology and fine structure of the giant interneurons of the wood cricket Nemobius sylvestris.

The structural and ultrastructural characteristics of giant interneurons in the terminal abdominal ganglion of the cricket Nemobius sylvestris were investigated by means of cobalt and fluorescent dye backfilling and transmission electron microscopy. The projections of the 8 eight pairs of the biggest ascending interneurons (giant interneurons) are described in detail. The somata of all interneurons analyzed are located contralateral to their axons, which project to the posterior region of the terminal ganglion and arborise in the cercal glomerulus. Neuron 7-1a is an exception, because its arborisation is restricted to the anterior region of the ganglion. The fine structure of giant interneurons shows typical features of highly active cells. We observed striking indentations in the perineural layer, enabling the somata of the giant interneurons to be very close to the haemolymph. The cercal glomerulus exhibits a high diversity of synaptic contacts (i.e. axo-dendritic, axo-axonic, dendro-axonic, and dendro-dendritic), as well as areas of tight junctions. Electrical synapses seem to be present, as well as mixed synapses. The anatomical organization of the giant interneurons is finally discussed in terms of functional implications and on a comparative basis.

Acoustic-induced motion of the bushcricket (Mecopoda elongata, Tettigoniidae) tympanum.

Bushcrickets have a tonotopically organised hearing organ, the so-called crista acustica, in the tibia of the forelegs. This organ responds to a frequency range of about 5-80 kHz and lies behind the anterior tympanum on top of a trachea branch. We analyzed the sound-induced vibration pattern of the anterior tympanum, using a Laser-Doppler-Vibrometer Scanning microscope system, in order to identify frequency-dependent amplitude and phase of displacement. The vibration pattern evoked by a frequency sweep (4-79 kHz) showed an amplitude maximum which would correspond to the resonance frequency of an open tube system. At higher frequencies of about 30 kHz a difference in the amplitude and phase response between the distal and the proximal part of the tympanum was detected. The inner plate of the tympanum starts to wobble at this frequency. This higher mode in the motion pattern is not explained by purely acoustic characteristics of the tracheal space below the tympanum but may depend on the mechanical impedance of the tympanum plate. In accordance with a previous hypothesis, the tympanum moves over the whole tested frequency range in the dorso-ventral direction like a hinged flap with the largest displacement in its ventral part and no higher modes of vibration.

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.

Early embryonic development of the head region of Gryllus assimilis Fabricius, 1775 (Orthoptera, Insecta).

We report our investigations on the embryonic development of Gryllus assimilis, with particular attention to the head. Significant findings revealed with scanning electron microscopy (SEM) images include: (1) the pre-antennal lobes represent the anterior-most segment that does not bear any appendages; (2) each of the lobes consists of central and marginal regions; (3) the central region thereof develops into the protocerebrum and the optic lobes, whereas the marginal region thereof becomes the anterior portion of the head capsule; (4) the initial position of the antennal segment is posterior to the mouth region; (5) appendage anlagen are transitorily present in the intercalary segment, and they later vanish together with the segment itself; (6) a bulged sternum appears to develop from the ventral surface of the mandibular, maxillary and labial segments. Embryonic features are then compared across the Insecta and further extended to the embryos of a spider (Araneae, Chelicerata). Striking similarities shared by the anterior-most region of the insect and spider embryos lead the authors to conclude that such comparison should be further undertaken to cover the entire Euarthropoda. This will help us to understand the embryology and evolution of the arthropod head.

The terminal abdominal ganglion of the wood cricket Nemobius sylvestris.

The abdominal cerci of the wood cricket, Nemobius sylvestris, are covered by a variety of hair-like sensilla that differ in length, thickness, and articulation. Fillings from the cercal nerves with cobalt chloride and fluorescent dyes revealed the projection of sensory axons into the terminal abdominal ganglion of the ventral nerve chain. Two projection areas on each side of the terminal abdominal ganglion midline could be identified: a posterior cercal glomerulus and an anterior bristle neuropil. Axons from some cercal sensilla ascend through the connectives to reach the metathoracic ganglionic mass. As their axons pass through each segmental abdominal ganglion, they project medial arborization. Cross-sections of the terminal abdominal ganglion and retrograde fills with cobalt chloride and fluorescent dyes from connectives revealed several small cells and seven pairs of giant ascending interneurons organized symmetrically. Giant somata are located contralateral to their axons (diameters between 20 and 45 mum). The cercal projections overlap extensively with the dendritic fields of the giant interneurons. In the terminal abdominal ganglion, we identified nine longitudinal tracts, two major tracts, and seven smaller ones. The functional implications of the neuranatomical organization of the system are discussed on a comparative basis.