SummarySynchrotron-generated X-rays provide scientists with a multitude of investigative techniques well suited for the analysis of the composition and structure of all types of materials and specimens. Here, we describe the properties of synchrotron-generated X-rays and the advantages that they provide for qualitative morphological research of millimetresized biological organisms and biomaterials. Case studies of the anatomy of insect heads, of whole microarthropods and of the three-dimensional reconstruction of the cuticular tendons of jumping beetles, all performed at the beamline ID19 of the European Synchrotron Radiation Facility (ESRF), are presented to illustrate the techniques of phasecontrast tomography available for anatomical and structural investigations. Various sample preparation techniques are described and compared and experimental settings that we have found to be particularly successful are given. On comparing the strengths and weaknesses of the technique with traditional histological thin sectioning, we conclude that Correspondence to: Oliver Betz. Tel: +0049 (0) 7071 2972995; e-mail: oliver. betz@uni-tuebingen.de synchrotron radiation microtomography has a great potential in biological microanatomy.

Insects are known to exchange respiratory gases in their system of tracheal tubes by using either diffusion or changes in internal pressure that are produced through body motion or hemolymph circulation. However, the inability to see inside living insects has limited our understanding of their respiration mechanisms. We used a synchrotron beam to obtain x-ray videos of living, breathing insects. Beetles, crickets, and ants exhibited rapid cycles of tracheal compression and expansion in the head and thorax. Body movements and hemolymph circulation cannot account for these cycles; therefore, our observations demonstrate a previously unknown mechanism of respiration in insects analogous to the inflation and deflation of vertebrate lungs.

In insects, anatomical features of the head have been found to provide important information for phylogenetic and comparative evolutionary studies. We analyzed the internal head morphology of three (omaliine, tachyporine, oxyteline group) out of the four subfamily groups of the beetle family Staphylinidae plus two non-staphylinid outgroups (i.e., Agyrtidae and Leiodidae). Synchrotron X-ray micro-tomography was used to obtain comparative head anatomical datasets of eight species to describe (i) the presence/absence of muscles inside the head capsule and (ii) the variability in their points of origin. Nineteen of these muscles were phylogenetically informative (nine with respect to presence/absence and eleven with respect to the origin; one muscle had an influence on both analyses) and were used in character mapping analyses to reconstruct groundplan patterns of the head musculature in Staphylinidae and their subgroups. Three muscles (Mm. 7, 9, 50) were identified as possibly autapomorphic for Staphyliniformia. The taxon (Agyrtidae + Leiodidae) is supported by the absence of M. 9. The monophyly of the tachyporine group is supported by a common origin of M. 4. Aleocharinae, a subfamily within the tachyporine group, is supported by the absence of M. 42 and possibly by numerous points of origin of various muscles (Mm. 1, 17, 28, 29, 30). Our analysis of the general organization of the hypopharynx-prementum-complex has revealed that this complex is organized in a similar way in the investigated staphylinoids, i.e., with the prementum lying anteriorly to and being in line with the hypopharynx and the mentum. We have found deviating conditions in the investigated species of the Aleocharinae, in which the prementum can be largely retracted posteriorly. Consequently, it is sandwiched between the ventral mentum and the dorsal hypopharyngeal region. The hypopharyngeal region is thus lifted dorsad to a large extent, approaching the cibarial roof. This situation is paralleled by a loss of the hypopharyngeal retractor (M. 42) and a shift of origin of premental retractors (Mm. 28-30) posteriorly toward the gula.

Abstract Betz, O., Thayer, M.K., and Newton, A.F. 2003. Comparative morphology and evolutionary pathways of the mouthparts in spore-feeding Staphylinoidea (Coleoptera). -Acta Zoologica (Stockholm) 84 : This study surveys the external morphology of the mouthparts in the guild of spore-feeders among the coleopterous superfamily Staphylinoidea, evaluating the influence of different phylogenetic and ecological starting points on the formation of their mouthparts. Our emphasis is on a scanning electron microscope analysis (SEM) of the involved trophic structures in spore-feeding larvae and adults of the Ptiliidae, Leiodidae and Staphylinidae, describing the fine structure of their main functional elements. Functionally, mouthpart structures resemble brushes, brooms, combs, rakes, rasps, excavators, knives, thorns, cram-brushes, bristle troughs, blocks and differently structured grinding surfaces. Their different involvement in the various aspects of the feeding process (i.e. food gathering, transporting, channelling and grinding) is deduced from our SEM analyses plus direct video observations. We infer five different patterns of food transport and processing, discriminating adults of ptiliids, leiodids plus staphylinids (excluding some aleocharines), several aleocharine staphylinids, and the larvae of leiodids and staphylinids. The structural diversity of the mouthparts increases in the order from (1) Ptiliidae, (2) Leiodidae towards (3) Staphylinidae, reflecting the increasing systematic and ecological diversity of these groups. Comparisons with non-spore-feeders show that among major lineages of staphylinoids, shifts from general microphagy to sporophagy are not necessarily constrained by, nor strongly reflected in, mouthpart morphology. Nevertheless, in several of these lineages the organs of food intake and grinding have experienced particular fine-structural modifications, which have undergone convergent evolution, probably in response to specialized mycophagy such as spore-feeding. These modifications involve advanced galeal rakes, galeal or lacinial 'spore brushes' with arrays of stout bristles, reinforced obliquely ventrad orientated prosthecal lobes and the differentiations of the molar grinding surfaces into stout teeth or tubercles. In addition, several staphylinids of the tachyporine and oxyteline groups with reduced mandibular molae have evolved secondary trituration surfaces, which in some aleocharines are paralleled by considerable re-constructions of the labium-hypopharynx.

SEM studies show that the differentiation among Stenus species with respect to the formation of the tarsi (wide bilobed vs. slender tarsomeres) takes place with a considerable augmentation of tarsal ventral setae in wide bilobed tarsomeres. The structural diversity of ventral tarsal setae among and within species is discussed with respect to 1) their different roles as mechanosensilla and tenent setae, respectively, and 2) the different selection pressures in terms of adhesive requirements along the longitudinal tarsus axis. The tarsi are provided with four groups of tarsal mechanosensilla, comprising hair and bristle sensilla, campaniform sensilla, and scolopidia. The tarsus wall is supported by an epidermis, which forms three different types of glands pouring their secretion via different exit paths onto the outer cuticle. The organization and ultrastructure of each of these glands is described. Only one (unicellular) gland is directly associated with the ventral tenent setae and is thus considered to form the main part of the adhesive secretion. The beetles appear to release the tarsal secretion through mediation of the tenent setae, which contains a lipid and a proteinaceous fraction. I propose that the secretion is discharged to the outside via a system of very fine pore canals in the wall of the setal shaft. Gas chromatography and infrared spectroscopy revealed that the lipid fraction of the secretion is a mixture of unsaturated fatty acid glycerides and aliphatic hydrocarbons whose spectra are similar to those of extractions of the superficial lipid coating of the body surface.

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To investigate whether specialization to spore- (or pollen-) feeding in advanced Aleocharinae is mirrored by their head anatomy, we compiled and compared synchrotron X-ray micro-tomography datasets for 11 Aleocharinae in conjunction with previous data for two aleocharine and six outgroup species (two nonstaphylinids, four staphylinids). We describe the presence/absence of head muscles and investigate the variability of points of origin by character mapping analyses. Monophyly of Aleocharinae is supported by the absence of M. 48 (M. tentoriobuccalis anterior), and by changes in the origins of Mm. 1, 2, 17, 18, 28, 29, 30. Within Aleocharinae the origins of the labial muscles (Mm. 28-30) have shifted posteriorly to the gula, which might enhance the movement posterad of the hypopharynx and partly compensate for the loss of M. 48. We also analyzed the general organization of the hypopharynx-prementum complex and the fine structure of the mandibles through SEM studies. In the absence of grinding mandibular molae like those of most mycophagous Coleoptera, seven aleocharine species studied have evolved "pseudomolae" at the ventral side of the mandibles that replace true molae as secondary grinding surfaces. In these species, the hypopharynx is elevated and displaced anteriorly, bearing a bowl-like depression on its surface that functions as a mortar where spores are ground between the hypopharynx and the mandibles. Two of these species are not yet known to feed on spores or pollen. Another species (Oxypoda alternans) is thought to feed on fungus material but bears no pseudomolae on its mandibles.

A literature survey is provided summarizing the available information on exocrine epidermal glands that produce adhesive secretions in insects. The focus is on both the ultrastructure of the gland cells and the identity and function of the chemical secretion produced by them. Insects employ adhesives for various functions such as tarsal attachment during locomotion, resisting external detachment forces, mating, phoresy and parasitism, egg anchorage, retreat building, self-grooming, prey capture, and active and passive defence. The available studies on the ultrastructure and the secretion of adhesive insect glands cover a broad spectrum of developmental stages and higher taxa, i.e.