Synchrotron X-ray Microtomography with Improved Image Quality by Ring Artifacts Correction for Structural Analysis of Insects.

Ring artifacts are undesirable and complicate the analysis and interpretation of microstructures in synchrotron X-ray microtomography. Here, we propose a new method to improve the image quality of an object by removing the ring artifacts and investigate the efficiency of this process with tomographic images of a dried Tenebrio molitor. In this method, before the tomographic reconstruction, ring artifacts were identified and located in the sinograms as line artifacts. Then, the identified line artifacts were corrected as single point noise via image processing of the original projections. Eventually, the corresponding line artifacts were removed, resulting in reduced ring artifacts in the reconstructed tomographic images. Simulations verified the efficiency of the proposed method. This method was successfully applied for the structural analysis of the insect T. molitor, showing superior performance in reducing ring artifacts in the tomographic image without noticeable loss of structural information.

Testicular and spermatogenic characteristics of Lagria villosa (Tenebrionidae: Lagriinae) with taxonomic inferences.

In the present study testicular and spermatogenetic aspects were described for Lagria villosa using light and scanning electron microscopy. In this species, spermiogenesis results in the formation of sperm bundles with spermatozoa arranged in an antiparallel manner, a characteristic observed only in Tenebrionidae. L. villosa, however, has about 60 follicles per testis and up to 1200 spermatozoa per cyst, in contrast to other tenebrionids that exhibit only six follicles in each testis and up to 512 spermatozoa per cyst. Therefore, the antiparallel arrangement of the spermatozoa in the bundle give support to previous works classifying the lagriids in a subfamily (Lagriinae) of Tenebrionidae. Just as the number of spermatozoa per cyst and follicles per testis suggest that they constitute, in fact, a distinct branch of this family.

[Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

Effect of ecdysone agonist RH-0345 on reproduction of mealworm, Tenebrio molitor.

RH-0345 belongs to a new group of insect growth regulators (IGRs) with a benzoylhydrazine structure that mimic the action of the natural insect molting hormone 20-hydroxyecdysone. After topical application on female adult beetles of mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), first oviposition was delayed, the number of eggs per female was reduced by 32%, the follicular epithelium was thinner (-33%) during sexual maturation, the size of deposited eggs was reduced, and egg viability was lost by 68%. Treatment with RH-0345 had also reduced the ovarian protein content and two protein bands were missing in the ovaries. Ultrastructural observations of the ovaries at the end of vitellogenesis in treated females, however, showed no evident differences with the fine structure of both follicular cells and oocytes in controls. In addition, we measured the amount of ecdysteroids in the medium of treated ovary cultures in vitro and in the eggs deposited by treated females. Possible action sites with the reproductive system at different levels in T. molitor are discussed for this novel group of IGRs.

[Cajal bodies in insect oocytes. I. Identification and immunocytochemical characteristics of Cajal bodies in vitellogenic oocytes of the yellow mealworm beetle]. / Tel'tsa Kakhala v ootsitakh nasekomykh. 1. Identifikatsiia i immunotsitochimicheskaia kharakteristika telest Kakhala v vitellogennykh ootsitakh zhuka-chernotelki.

In vitellogenic oocytes of Tenebrio molitor (inactive stage), numerous fibrogranular nuclear bodies (NBs) are present. Using immunofluorescent microscopy, these NBs were shown to contain pre-mRNA splicing factors (small nuclear [sn] RNPs and SR-protein, SC35) as well as RNA polymerase II. A limited set of NBs also contained coilin, a marker protein for Cajal bodies (CBs). We suggest that in T. molitor oocytes, coilin-containing NBs, which also contain splicing factors and RNA polymerase II, seem to represent CBs. In the species studied, no morphological features of CBs were established as compared with other NBs, which do not contain coilin. Microinjectons in oocytes of myc-tagged coilin mRNA, followed by revealing newly translated protein with antibody specific for this tag, have shown targeting of myc-coilin with CBs. The own and literary data on the morphology and molecular composition of CBs are discussed in terms of searching for criteria for CB identification in cells of different origin, and at active and inactive stages.

Molecular cloning and functional properties of two early-stage encapsulation-relating proteins from the coleopteran insect, Tenebrio molitor larvae.

Encapsulation is a major defensive reaction against foreign materials that are too large to be phagocytosed by individual hemocytes; however, the biochemical process of encapsulation is still obscure. To isolate and characterize the early-stage encapsulation-relating protein (ERP), we used the coleopteran insect, Tenebrio molitor larvae, injecting three differing kinds of bead or inserting pieces of surgical suture into the abdomen of T. molitor larvae. The resulting proteins from the injected beads or the inserted pieces of surgical suture were recovered 10 min after injection or insertion, and were analyzed on SDS/PAGE under reducing conditions. Four different proteins (86, 78, 56 and 48 kDa) were enriched compared with the crude hemolymph. Among them, we purified 56-kDa and 48-kDa ERPs to homogeneity and raised polyclonal antibodies against each protein. Immunoblotting analysis showed that the affinity-purified antibodies of the 56-kDa and 48-kDa ERPs cross-reacted with the 48-kDa and 56-kDa ERPs, respectively. Analysis of the cDNA of 56-kDa ERP consisted of 579 amino acid residues and showed a novel glutamine-rich protein. Positive clones of the 48-kDa ERP showed the same DNA sequence as 56-kDa ERP. Interestingly, the chemically determined N-terminal amino acid sequence and the three partial amino acid sequences of the 48-kDa protein were found in the 56-kDa ERP, suggesting that the 48 kDa ERP was produced by the cleavage of Arg101-Gly102 of the 56-kDa ERP by a limited proteolysis. Western blotting analysis showed that these ERPs were detected exclusively on membrane fractions of hemocytes. Also, when the early-stage encapsulated beads were coated with both the 56-kDa and 48-kDa ERP antibodies and re-injected into larvae, no further encapsulation reaction was observed. However, when the early-stage encapsulated beads were incubated with 56-kDa ERP antibody, 48-kDa ERP antibody or nonimmunized rabbit IgG and re-injected into larvae, further encapsulation did occur.

Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period in Tenebrio molitor.

In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.

Microtubule organization and the distribution of gamma-tubulin in spermatogenesis of a beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta).

The present study focuses on the restructuring of the microtubule (MT) cytoskeleton and microtubule-organizing centres (MTOCs) throughout spermatogenesis of a darkling beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta). To this end, serial ultrathin sections through male germ cells were studied using transmission electron microscopy. Additionally, spindles and young spermatids were isolated from testes under MT-stabilizing conditions and doubly labeled with antibodies against beta- and gamma-tubulin. The latter is a tubulin isoform detected in MTOCs of a wide variety of species. The observations suggest that microtubules may be nucleated from sites with and without high gamma-tubulin content and that these sites do not necessarily possess canonical centrosomes. In a prominent cytoplasmic MT system of primary spermatocytes in prophase, microtubule nucleation apparently occurs in the absence of immunologically detectable gamma-tubulin. At the poles of meiotic spindles, MTs are directly inserted into gamma-tubulin-containing material and this connection is considered responsible for their nucleation. The interzone spindle MTs of telophase cells contain gamma-tubulin and this may confer stability to them. Finally, manchette MTs of spermatids originate in the vicinity of the acrosome precursor but are not inserted into this body. The acrosome precursor is surrounded by a membrane and is clearly detected by the antibody against gamma-tubulin.

Intracellular dye injection of previously immunolabeled insect neurons in fixed brain slices.

Our method combines intracellular dye injection and immunohistochemistry. Under optical control, Lucifer Yellow was injected into immunohistochemically identified neurons that reside in fixed tissue. The technique allows visualization of the complete arborization patterns of immunostained neurons. Injections were performed on small neurons (somata < 10 microns in diameter). The technique works on microslices of insect brain. Standard immunohistochemical procedures have only been varied slightly, omitting Triton X-100 treatment. Anti-Lucifer Yellow immunohistochemistry, or alternatively the photoconversion technique, enables extension of the morphological analysis of these cells to the electron microscopic level. In the present study, Lucifer Yellow injections were performed on immunohistochemically pretreated brain microslices (anti-Locusta tachykinin II antiserum) of the beetle Tenebrio molitor.

Filipin-cholesterol complexes in plasma membranes and cell junctions of Tenebrio molitor epidermis.

The polyene antibiotic filipin combines with cholesterol in membranes to form complexes that are readily identifiable in the electron microscope. The distribution of filipin-cholesterol (FC) complexes is most easily studied by freeze-fracture. Larval epidermis of Tenebrio molitor (Insecta, Coleoptera) was maintained in vitro for 48 hr, since the electrophysiological properties of the cells are best characterized under these conditions. The cells were fixed in buffered 3.0% glutaraldehyde at RT for 15 min, transferred to fresh fixative containing 1% DMSO and filipin (final concentration; 0.5 mg/ml) for 3 hr RT. Control cells were treated in fixative containing 1% DMSO only. In freeze fracture replicas, FC complexes appear on the plasma membrane as large circular protrusions measuring 26.5 +/- 6.8 nm (x +/- s.d.) n = 50, in diameter and 17.1 +/- 2.8 nm, n = 50, in height and 11.7 +/- 2.6 nm, n = 25, in depth. Protrusions are about two times more frequent on the E face while pits are several times more frequent on the P face. FC complexes are most abundant (greater than 50/mu m2) on the basal membrane surface of the cells but are excluded from regions of hemidesmosomal plaques that anchor the cells to the basal lamina. FC complexes are also abundant on the apical surfaces of the cells where cuticle secretion occurs. In the lateral regions below the junctional belt, FC complexes are less numerous but often appear to increase in frequency in a graded fashion away from the junctional region. The septate junctions are relatively free of FC complexes except in regions where they open to form islands. These islands often contain gap junctions but the FC complexes rarely invade the particle domains of the gap junctions. Single FC complexes were seen in three out of a total of 97 gap junctions. Exposure of the epidermis to 20-hydroxyecdysone for 24 hr in vitro did not induce the appearance of FC complexes within the cell junctions.

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes.

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl2, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM-20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM-100 mM NaCl, 0.2 mM-1.0 mM MgCl2). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl2 concentrations up to 0.4 mM. In higher MgCl2 concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl2 and of 65-nm-thick fibers in 1.0 mM MgCl2. After the 37-nm and the 65-nm fibers had been dispersed in Mg2+-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl2, respectively. It is concluded that a Mg2+-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.

Isolation and characterization of invertebrate smooth septate junctions.

Using modifications of techniques used for the isolation of macula type intercellular junctions (gap junctions and desmosomes) the arthropod smooth septate junction has been isolated from insect midgut tissue. Midguts from cockroaches or mealworms were used and membrane fractions were obtained by sucrose gradient and ultracentrifugation techniques. Preparations with reasonable concentrations of septate junction were obtained and have been studied by thin-section, negative-stain and freeze-fracture electron microscopy. The junctions appeared to be well preserved, although there was evidence that the junction strands were able to slide within the plane of the membrane. Septa were seen to have a cross-striated appearance when viewed after negative staining but their exact structure remained difficult to determine. Polyacrylamide gel electrophoretic studies demonstrated the reproducibility of the isolation procedure and showed that septa may have a 47 000 molecular weight glycoprotein component. Gel electrophoresis also gave some indication of the intramembrane biochemistry of the smooth septate junction, with proteins of 31 000 and 32 000 molecular weight always occurring in the junction fractions. The junctions were, however, very sensitive to both mechanical and chemical treatments, the septa were destroyed by rough homogenization or by treatment with urea at a concentration as low as 1 M. Freeze-fracture of untreated, isolated junctions demonstrated no differences from junctions in intact tissue, while replicas of urea-treated material were more difficult to interpret as the component parts of the junctions became separated once the septa had been destroyed. Gap junctions were also obtained and resisted both mechanical and chemical treatment, which destroyed the septate junctions. Their major protein component appeared to have a molecular weight of 36 000. Attempts to isolate pleated septate junctions (from insects, molluscs and annelids) by the same techniques failed, implying a significant difference in the structures of the two types of septate junction.

The effect of temperature on muscle resting potential in some insects.

The effect of temperature on muscle resting potential was studied in Acheta domesticus, Leptinotarsa decemlineata and Tenebrio molitor. The experiments were performed using the conventional microelectrode method and specific physiological solution for each insect species. The measurements were taken at three temperature levels: +/- 4 degrees C, +/- 20 degrees C and +/- 35 degrees C. Placing the preparations into a bath at 4 degrees C caused in all three species a rapid decrease of the muscle resting potential (+/- by 1/3). Increased temperature (+/- 35 degrees C) led to a transient increase of the resting potential (10 min after placing in the solution), and then a decrease of the potential below the control values. The possible mechanisms of these findings based both on ionic gradients, permeabilities and metabolic activity are discussed.

Ultrastructural comparison of the perisympathetic organs in three Coleoptera: Chrysocarabus auronitens F., Oryctes rhinoceros L. and Tenebrio molitor L.

Ultrastructural comparison of different types of perisympathetic organs (POs) in three species of Coleoptera (Chrysocarabus auronitens, Oryctes rhinoceros, and Tenebrio molitor) showed that the structure of these organs was not related to their morphological types but to their topography. Two kinds of PO structure may be distinguished: compact median and diffuse lateral. They were similar in that both were surrounded by thin neural lamellae and exhibited numerous glial cells originating in the perineurium (type I perineurial cells) as well as abundant neurosecretory endings. They were different in as much as in median POs, the neurosecretory endings were generally surrounded by perineurial processes but in transverse POs, these endings were sheathless. Only one type of neurosecretory axon was distinguished in the median organs but three or four in the transverse. The nature of the processes by which neurosecretory granules are released may depend on the type of neurosecretory axon. For instance, exocytosis always occurred for dense spherical granules, and granule fragmentation was visualized for granules of smaller size.

The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera).

In order to study the pupal-adult metamorphosis of Tenebrio in vitro, pupal sternites of different ages were cultured in Landureau's medium and their development systematically observed by electron microscopy. In hormone-free medium, explants taken from young pupae do not secrete pupal postecdysial cuticle in vitro, and the epidermis spontaneously detaches from the pupal cuticle. On the contrary, explants taken from pharate adults continue to secrete adult preecdysial cuticle in vitro, and the epidermis never detaches from the cuticle. Ecdysterone in physiological concentrations (0.2 to 4 micrograms/ml) induces the secretion of a new cuticle in explants from young pupae but the epidermis remains undifferentiated. Ecdysone is necessary for the induction of some adult differentiation. Moreover, the quality of the cuticle secreted in vitro is increased by the addition of 2% foetal calf serum; the best results have thus far been obtained in a medium containing 0.2 microgram/ml ecdysone, 1 microgram/ml ecdysterone, and 2% foetal calf serum.

The supply of oxygen to the flight muscles of insects: a theory of tracheole physiology.

In the flight muscles of insects, virtually every mitochondrion is in contact with or is encircled by terminal tracheoles which reach them by following the channels formed by the invaginated plasma membrane of the muscle fibres, the T-system tubules. In musca, Calliphora and Drosophila (Diptera), Apis (Hymenoptera) and Tenebrio (Coleoptera) the terminal tracheoles are smooth-surfaced tubes with a lumen of about 50 nm. In Pieris (Lepidoptera) the terminal tracheoles occupy the regular transverse tubular system which runs between the mitochondria and across the fibrils on either side of the H zone. They are smooth tubules of 80-200 nm diameter. Preliminary observations suggest the same arrangement in Ischnura (Odonata). In Rhodnius and other Hemiptera the transverse T-tubule system forms large cavities among the mitochondria: these cavities in Rhodnius are occupied by smooth-walled tracheole endings. In the nature adult of Schistocerca (Orthoptera) T-tubules of varying size are utilized by terminal tracheoles (diameter 50-100 nm). The terminal tracheoles of the flight muscles are highly permeable to myrcene and kerosene. They commonly fill with liquid during rest and this liquid is resorbed during activity. It is suggested that these adaptations increase the efficiency of respiration in the flight muscles by ensuring that, when it is most needed, gaseous oxygen extends to the surface of the mitochondria, from which it is separated by a very permeable barrier.

Observations on cytoplasmic transport along ovarian nutritive tubes of polyphagous coleopterans.

The ovarioles of Coccinella and Tenebrio are shown to be telotrophic--a characteristic normally associated with hemipterans rather than coleopterans. The possess an anterior region of trophic cells and a chain of oocytes. The trophic cells are connected with the latter by a series of nutritive tubes, and autoradiography has shown that RNA is transported along the tubes to the oocytes. However, the system in these beetles differs markedly from that of hemipterans in that the nutritive tubes do not contain an extensive complement of aligned microtubules. The significance of this to both the mechanism and the selectivity of transport is discussed.