The Last Breath: A µCT-based method for investigating the tracheal system in Hexapoda.

In recent years, µCT-based studies of the insect tracheal system have become an increasingly important area of research. Nevertheless, the methods proposed in previous research for investigating the respiratory system in the three-dimensional space were described and tested based on a relatively small number of specimens. Additionally, the individuals studied in all these cases represented only a single post-embryonic stadium - pupa or imago - of a particular insect species. Therefore, in the current situation it is difficult to predict the reliability and possible limitations of these methods. To address this problem we conducted a methodological study, during which we used 65 individuals representing larvae, pupae and imagines of the mealworm beetle (Tenebrio molitor). In addition to the protocol previously described, which implicated freezing as a killing technique, we also tested a novel one, which was based on ethyl acetate fumigation of the specimens studied. We included step-by-step guides for the manual and semiautomatic approaches in order to facilitate the digital visualization of the tracheal system. Our investigations enabled us to generate multiple models of the tracheal system of all post-embryonic stages of the mealworm beetle. The methods used proved to be minimally invasive, thus allowing for the application of post-scanning manipulations, such as drying with critical point dryer (CPD). This approach enabled us to merge different three-dimensional models into a single picture and analyse the relationship of the tracheal system with other tissues (e.g., muscles, nervous system). We comprehensively discuss the advantages and possible limitations of the tested methods and provide practical suggestions for conducting the analyses on a wider scale. The visualizations presented in this publication are the first three-dimensional models of the respiratory system using a representative of the extremely diverse order Coleoptera.

Insect morphological diversification through the modification of wing serial homologs.

Fossil insects living some 300 million years ago show winglike pads on all thoracic and abdominal segments, which suggests their serial homology. It remains unclear whether winglike structures in nonwinged segments have been lost or modified through evolution. Here, we identified a ventral lateral part of the body wall on the first thoracic segment, the hypomeron, and pupal dorsolateral denticular outgrowths as wing serial homologs in the mealworm beetle Tenebrio molitor. Both domains transform into winglike structures under Hox RNA interference conditions. Gene expression and functional analyses revealed central roles for the key wing selector genes, vestigial and scalloped, in the hypomeron and the denticular outgrowth formation. We propose that modification, rather than loss, of dorsal appendages has provided an additional diversifying mechanism of insect body plan.

Morphological correlates of a combat performance trait in the forked fungus beetle, Bolitotherus cornutus.

Combat traits are thought to have arisen due to intense male-male competition for access to females. While large and elaborate weapons used in attacking other males have often been the focus of sexual selection studies, defensive traits (both morphological and performance) have received less attention. However, if defensive traits help males restrict access to females, their role in the process of sexual selection could also be important. Here we examine the morphological correlates of grip strength, a defensive combat trait involved in mate guarding, in the tenebrionid beetle Bolitotherus cornutus. We found that grip strength was repeatable and differed between the sexes. However, these differences in performance were largely explained by body size and a non-additive interaction between size and leg length that differed between males and females. Our results suggest that leg size and body size interact as part of an integrated suite of defensive combat traits.

Body size is not critical for critical PO2 in scarabaeid and tenebrionid beetles.

Constraints on oxygen delivery potentially limit animal body size. Because diffusion rates are highly distance dependent, and because tracheal length increases with size, gas exchange was traditionally thought to be more difficult for larger insects. As yet the effect of body size on critical oxygen partial pressure (P(crit)) has not been measured for any clade of insect species for which there are interspecific data on tracheal scaling. We addressed this deficiency by measuring P(crit) over a 4150-fold mass range (ratio of largest to smallest species mean) of two families of Coleoptera (Tenebrionidae and Scarabaeidae). We exposed adult beetles to progressively lower oxygen levels and measured their ability to maintain CO(2) release rates. Absolute metabolic rates increased hypometrically with beetle body mass (M) at both normoxic (M(0.748)) and hypoxic (M(0.846)) conditions. P(crit), however, was independent of body size. Maximum overall conductances for oxygen from air to mitochondria (G(O(2),max)) matched metabolic rates as insects became larger, likely enabling the similar P(crit) values observed in large and small beetles. These data suggest that current atmospheric oxygen levels do not limit body size of insects because of limitations on gas exchange. However, increasing relative investment in the tracheal system in larger insects may produce trade-offs or meet spatial limits that constrain insect size.

[Effect of weak combined magnetic fields on the metamorphosis of the meal-worm beetle Tenebrio molitor].

The effects of weak combined magnetic fields adjusted to the parametric resonance for Ca2+ and K+ and extremely weak alternating magnetic field on the metamorphosis of the meal-worm beetle Tenebrio molitor have been studied. It was shown that the exposure of pupas of insects to all above-indicated types of fields stimulates the metamorphosis. However, after the exposure to weak combined magnetic fields adjusted to the parametric resonance for Ca2+ and K+, the number of insects with anomalies increases, which is not observed by the action of the weak alternating magnetic field.

New species of the South American loxandrine genus Metoncidus Bates (Coleoptera, Carabidae)

Duas espécies novas são acrescentadas ao gênero, anteriormente monotípico, Metoncidus Bates, 1871 (Carabidae, Loxandrini): M. epiphytus sp. nov. (localidade-tipo Peru: Loreto, Cocha Shinguito) e M. gracilus sp. nov. (localidade-tipo Peru: Tambopata, Madre de Dios). Informações que permitem a identificação do gênero dentre outros gêneros sul-americanos de carabídeos e uma chave para espécies de Mentocidus são fornecidas.

Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein in Tenebrio molitor.

The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hybridization analysis showed that ACP17 mRNA expression begins weakly 3 days before adult ecdysis and strongly increases during the secretion of postecdysial adult cuticle, with a maximum just after ecdysis. In situ hybridization revealed that the ACP17 mRNA is only present in the epidermis which secretes hard cuticle. The deduced amino acid (aa) composition exhibits a high content of Gly (28%) and Ala (20%) and, particularly, two poly(Gx) stretches separated by repetitive motifs with proline AAPVA. A comparison is made with other cuticle aa sequences.

cDNA-inferred amino-acid sequence of a C protein, a heparin-binding, basic secretion product of the tubular accessory sex glands of the mealworm beetle, Tenebrio molitor.

C proteins represent one of the four major protein groups secreted by the tubular accessory glands of male mealworm beetles (Tenebrio molitor). They are basic proteins with an apparent molecular mass of 21.9 kDa. In this paper we present the deduced amino-acid sequence of two, almost identical C proteins, termed C1 and C2. The C proteins contain a consensus sequence for a heparin-binding site, and they are efficiently purified from accessory gland homogenates by heparin-affinity chromatography. No sequence resemblance was found with other proteins in the databases, but their high avidity for heparin suggests a possible involvement of the C proteins in sperm capacitation.

Crustacean cardioactive peptide-immunoreactive neurons in the hawkmoth Manduca sexta and changes in their immunoreactivity during postembryonic development.

An antiserum against crustacean cardioactive peptide was used, in indirect immunocytochemistry on whole-mounts and Vibratome sections, to map immunoreactive neurons at various stages of postembryonic development of the hawkmoth Manduca sexta. About 90 immunoreactive neurons were identified. Many of these cells are immunoreactive at hatching and persist into the adult stage; others become immunoreactive late in postembryonic development. During adult development, transient immunoreactivity is expressed in several cells in the subesophageal and thoracic ganglia. Two sets of immunoreactive neurons are found in the protocerebrum of larvae, but only one of these sets persists into the adult stage. Paired lateral interneurons and neurosecretory neurons are segmentally repeated in the abdominal ganglia and are present from the first larval stage to the adult; the abdominal interneurons project contralaterally to arborizations in adjacent ganglia, and some ascend to tritocerebral arborizations. The abdominal neurosecretory cells, which correspond to a pair of cells reported to contain bursicon, project posteriorly to neurohemal release organs. Motor neurons of dorsal external oblique abdominal muscles become immunoreactive in the fourth larval stage. Paired median neurosecretory cells of abdominal ganglia become immunoreactive during the fifth larval stage. The immunoreactive median and lateral abdominal neurosecretory cells are a subset of a group of cells known to contain cardioactive peptides. Paired lateral neurosecretory cells of the subesophageal ganglion become immunoreactive during pupation and project to the corpora cardiaca and aorta of the adult. Many of the neurons identified here are comparable to crustacean cardioactive peptide-immunoreactive cells described previously in locusts and the mealworm beetle.

Fate of descending interneurons in the metamorphosing brain of an insect, the beetle Tenebrio molitor L.

The fate of descending brain-thoracic neurons in the metamorphosing supraoesophageal ganglion of the mealworm beetle, Tenebrio molitor, is described. Comparison of the descending neurons of the larval, various pupal, and adult stages outlines a high degree of topological invariance in the structure of descending interneurons; i.e., the basic organization of the imaginal set of descending neurons is anticipated by the structure of the larval neurons. Single descending neurons of analogous clusters of larval and imaginal neurons outline a virtual identical structural organization in both the larval and imaginal brain. There is a quantitative increase from approximately 70 to approximately 120 brain-thoracic interneurons during metamorphosis. This increase does not cause complex transformations in the structural organization of the descending interneurons. Experiments with the DNA-inhibitor hydroxyurea prove that the described topological invariant structure of the set of descending neurons is based on the persistence of individually descending neurons. There is evidence that the whole set of larval interneurons persists throughout the beetles' metamorphosis. The essential characteristics of the later imaginal set of descending neurons are qualitatively and quantitatively established within the first 10% of pupation. Structural invariance of the set of descending neurons is discussed with regard to the significance of cellular interaction for the mechanisms of metamorphic reorganization of nervous tissue.

Ecdysteroids in vitro promote differentiation in the accessory glands of male mealworm beetles.

Physiological peak doses of 20-hydroxyecdysone were added to organ cultures of young pupal accessory glands of male Tenebrio molitor. During subsequent culture in vitro or in vivo, the glands accumulated adult-specific antigens. Control organ cultures showed no such antigen accumulation. In this system, ecdysteroid controls not only cell cycles but also differentiation.

Oxygen-17 compounds as potential NMR T2 contrast agents: enrichment effects of H2(17)O on protein solutions and living tissues.

The isotopic enrichment of solutions, living tissues, and organisms with oxygen-17 in the form of H2(17)O shortens their proton NMR transverse relaxation times (T2) and produces changes in NMR image intensity. The transverse relaxation rate (1/T2) was found to be linearly dependent on the H2(17)O concentration in biological solutions up to 5% enrichment. The longitudinal relaxation time (T1) is not affected by enrichment. Equal concentrations of H2(17)O do not produce the same magnitude of T2 change in all physiological environments. The reasons for these differences are discussed. The results suggest that certain oxygen-17 compounds should be explored as "contrast agents" in magnetic resonance imaging.