Role of 24- and 28-hydroxylated intermediates in the metabolism of beta-sitosterol in the insect Tenebrio molitor.

1. [28-3H]Stigmast-5-ene-3 beta, 28-diol and [23,23,25-3H]stigmast-5-ene-3 beta, 24-diol were synthesized. 2. Each of the samples was mixed with beta-[4-14C]sitosterol and administered to Tenebrio molitor larvae. 3. The former compound is not utilized by the insect; the latter, although metabolized to 24(28)-ethylidene sterols and cholesterol, is not a beta-sitosterol metabolite. 4. The above results are discussed in relation to the mechanism of formation of the 24(28)-double bond in beta-sitosterol metabolism in T. molitor.     

Synthesis, cardiostimulatory, and cardioinhibitory effects of selected insect peptides on Tenebrio molitor

The subject of these studies was a search for proctolin antagonists among peptides originating from insect species because the proctolin antagonists constantly pose a problem. During these studies we performed the synthesis of the following peptides: a native decapeptide from Manduca sexta Mas-MT-I and its 11 analogs with shortened sequences at the N-end as well as a growth suppressor, a pentapeptide isolated from Antheraea yamamai, Any-GS and its 10 analogs, modified at position 1 and with a shortened peptide chain.Biological effects were evaluated by the cardiotropic test on the semi-isolated heart of the insect species Tenebrio molitor. Mas-MT-I and six analogs stimulate the heartbeat frequency, especially [6-10]-Mas-MT-I, whereas the [4-10]-Mas-MT-I analog shows a strong inhibition of the heartbeat frequency, if insect. The Any-GS and the analogs [Gln(1)]- and [Gly(1)]-Any-GS also show a strong cardioinhibitory effect.     

Primary and secondary structure of the 18 S ribosomal RNA of the insect species Tenebrio molitor

The sequence of the 18 S rRNA of Tenebrio molitor is reported. A detailed secondary structure model for eukaryotic small subunit rRNAs is proposed. The model comprises 48 universal helices that eukaryotic and prokaryotic small subunit rRNAs have in common, plus a number of helices in areas of variable secondary structure. For the central area of the model, an alternative structure is possible, applicable only to eukaryotic small subunit rRNAs. Possibly, small subunit rRNA switched to this alternative conformation after the eukaryotic branch had been established in evolution. Another possibility is that the two conformers represent a dynamic structural switch functioning during the translational activity of the eukaryotic ribosome.     

Structure and development of the larval central complex in a holometabolous insect,the beetle Tenebrio molitor

Summary The neuroarchitecture of the central complex, a prominent neuropil in the midbrain of the holometabolan, Tenebrio molitor, is described throughout larval development. The analysis is based on classical silver impregnations and on fate-mapping of identified neurons using antisera against serotonin and FMRF-amide. In T. molitor, the central body is present in the first larval instar, and is formed by side branches of contralaterally projecting neurons. Glial cells surround eight neuropil compartments in the first larval instar. These subdivisions in the organization of the fan-shaped body are maintained throughout development. Intrinsic interneurons are found from the 5th larval instar onwards. In the last larval stage, the central complex consists of the fan-shaped body, the protocerebral bridge, and the anlage of the ellipsoid body. The cellular architecture of the fan-shaped body of the last larval instar resembles the basic structural characteristics of the adult. Serotonin-immunoreactive neurons and FMRF-amide immunoreactive neurons in the midbrain of the first larval instar show the basic structural features of the respective imaginal cells. The structural organizations of larval and adult midbrain are compared.Abbreviations a Anterior - AGT antenno-glomerular tract - aL -lobus - AL antennal lobe - AP anterior protocerebrum - bL -lobe - BSN bilateral symmetrical - FMRF amide-immunopositive neurons - CA calyx - CL1-CL4 serotonin-immunopositive neurons cluster 1–4 - d dorsal - DAB diaminobenzidine tetrahydrochloride - DC dorsal commissure - DCFB dorsal commissure of the fan-shaped body - DHT dorsal horizontal tract - DLTR dorsal lateral triangle - DMLP dorsal medial lateral protocerebrum - DN serotonin-immunopositive deuterocerebral neuron - EB ellipsoid body - en1, en2 extrinsic neurons connecting two FB-subcompartments - esn extrinsic subcompartmental neuron - l lateral - FB fan-shaped body - FN serotonin-immunopositive fan-shaped neuron - fs1, fs2 fanshaped neurons of type 1 and 2 - GC great commissure - HF horizontal fibres - in intrinsic neuron connecting two FB-subcompartments - isn intrinsic subcompartmental neuron - IT isthmus tract - LF large-field neurons - LFASC lateral fascicle - LMFASC lateral median fascicle - MB median bundles - MLP medial lateral protocerebrum - p posterior - P pedunculus - PB protocerebral bridge - pb-fb protocerebral bridge-fan-shaped body connection - PBS phosphate-buffered saline - PDC posterio-dorsal commissure - PTX phosphate-buffered saline containing Triton X-100 - SU suboesophageal ganglion - SVT small ventral triangles - TN 1,2 tritocerebral serotonin-immunoreactive neuron 1,2 - v ventral - VB ventral body - VBC ventral body commissure - VCBC ventral central body commissure - VCFB ventral commissure of the fan-shaped body     

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.     

Host stadium specificity in the gregarine assemblage parasitizing Tenebrio molitor.

Reciprocal cross-stadia experimental infections were used to demonstrate stadium specificity within the gregarine assemblage parasitizing Tenebrio molitor, the yellow mealworm. Gregarina cuneata, Gregarina polymorpha, and Gregarina steini are characteristic parasites of larval T. molitor. Gregarina niphandrodes is a characteristic parasite of adult T. molitor. Experimental infections were produced in all homologous host-parasite combinations. No infection was produced in heterologous or cross-stadia combinations. This study introduces the concept of separate, distinct parasite niches corresponding to separate life cycle stages and established by known, predictable life cycle events within a single host species.     

Screening for mycotoxins with larvae of Tenebrio molitor.

Larvae of the yellow mealworm, Tenebrio molitor, were used to screen isolates of field and storage fungi, included in their dietary substrate, for mycotoxins. Feeding of three isolates of Fusarium roseum, two of Fusarium equiseti and of Fusarium nivale, and one of an unidentified species of Fusarium resulted in growth depression of the larvae. One isolate of an unidentified species of Myrothecium was also toxic to larvae of Tenebrio molitor. Mycotoxin production was apparently dependent, not only on the fungal isolate, but also on the culture conditions under which the fungus was grown. Some fungal isolates had growth-promoting qualities for larvae of Tenebrio molitor.     

Cytodifferentiation of the accessory glands of Tenebrio molitor

Summary Spermathecal accessory glands from pupae of Tenebrio molitor were cultured in vitro in Landureau S-20 medium with or without ecdysterone at a concentration of 5 g/ml medium. Morphological changes were examined by electron microscopy. Tissue taken from pupae that have not been exposed to a peak of ecdysterone in vivo is only able to differentiate in medium with hormone, and then only partially, while tissue taken from pupae that have experienced an endogenous peak of ecdysterone is able to develop maximally irrespective of the presence or absence of hormone. The specific ultrastructural changes that occur in vitro correspond to those occurring in the gland in situ during the normal course of differentiation, and are: the formation of the pseudocilium, cell retraction and formation of an end apparatus, and cuticulogenesis in the ductule and main lumen of the gland. Pseudocilium formation does not appear to be ecdysterone-dependent, while cuticulogenesis requires ecdysterone for initiation of the process. Deposition of cuticle is an expression of an earlier commitment of the cells to this process, and is initiated by elevated levels of ecdysterone. Ecdysterone is not required for completion of cuticulogenesis.This study was supported by grants from the National Institutes of Health (NIAMD 15662 and NIGMS 26140)     

Head movements of the mealworm beetle Tenebrio molitor

Tethered walking imagines of the mealworm beetle Tenebrio molitor wave their heads in random fashion. If a periodic pattern of vertical black and white stripes is rotated around the animal a regular nystagmic head movement is superimposed upon the random waving, the frequency of the latter equals the contrast frequency within large ranges of the angular velocity of the pattern. The nystagmus is inverted: After a short period of tracking, during which the angular velocity of the head is the same as that of the panorama, the head returns slowly toward its normal position according to an exponential-like function. Resting animals do not wave their heads. However, if the above panorama is rotated, the beetle turns its head in the direction of the movement of the panorama and holds it in a side-way position, as long as the rotation is maintained. The angular position reached depends in the same manner on the angular velocity of the panorama as the turning tendency of walking animals established in open loop experiments using the spherical Y-maze method.     

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.     

Sequence-induced curvature of Tenebrio molitor satellite DNA

Single satellite DNA constitutes about 50% of the Tenebrio molitor genome. Electrophoresis of 142 base pair long satellite monomers on nondenaturating polyacrylamide gel shows retarded mobility, a characteristic of fragments with sequence-induced DNA curvature. Migrational analysis of circularly permuted satellite monomers revealed the existence of 2 bend centers in the monomer sequence. We calculated the trajectory of DNA helix axis according to the algorithm of De Santis et al. This model predicts that T molitor naked satellite DNA forms a solenoid structure with left-handed superhelix. One turn of the superhelix has approximately 310 base pairs and a 33 nm pitch. Point mutations found in the satellite DNA (1.8%) influence bending characteristics, but do not distort the general geometry of satellite superhelix.