Incorporation of various putative precursors into cuticular 3,4-dihydroxyphenylacetic acid of adult Tenebrio molitor

Post-emergence levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and ketocatechol were determined in cuticle from adult Tenebrio molitor. Possible pathways for biosynthesis of DOPAC were studied by comparing the incorporation of injected [U-¹⁴C]tyrosine, [7-¹⁴C]dopamine, [7-¹⁴C]DOPA, [7-¹⁴C]tyramine, [U-¹⁴C]p-hydroxyphenylpyruvic acid (p-HPPA) and [ring-³H]p-hydroxyphenylacetic acid (p-HPAA) into cuticular DOPAC during its period of maximal increase 1–3 days after adult emergence. Increased incorporation of [U-¹⁴C]tyrosine between days 0 and 3 suggests rapid de novo biosynthesis of DOPAC from this primary precursor. Of the putative intermediates tested, only p-HPPA had a pattern of incorporation similar to that seen with tyrosine. Since p-HPAA was poorly incorporated into both cuticle and DOPAC, a tentative pathway tyrosine → p-HPPA → 3,4-dihydroxyphenylpyruvic acid → DOPAC is proposed.     

The cuticular proteins of Tenebrio molitor: I. Electrophoretic banding patterns during postembryonic development

Buffer-soluble cuticular proteins of the abdomen of the yellow mealworm, Tenebrio molitor, were analyzed by SDS-polyacrylamide gel electrophoresis. Since the abdominal epidermis of Tenebrio persists throughout the insect's life, these cuticular proteins reflect the secretory history of a continuous line of cells during its entire metamorphic developmental program. Twenty-two to thirty-eight bands were detected in extracts of larval cuticle, 11 to 35 in pupal cuticular extracts, and 30 to 41 in extracts from adults. No population polymorphism was apparent, nor was there any sexual dimorphism, in these cuticular proteins. At each metamorphic stage, the cuticular proteins formed a unique banding pattern. Bands unique to the larval and to the adult exocuticular extracts were observed. Extracts from cuticles of freshly ecdysed animals (exocuticle) differed from extracts from animals in which sclerotization and postecdysial (endocuticle) deposition had occurred, both in number of hands and in their molecular weight distributions. Some proteins became less soluble during sclerotization. The majority of the exocuticular bands from all three stages had molecular weights below 25,000; higher-molecular-weight proteins were extracted from postecdysial animals of each stage.     

The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.     

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta,Coleoptera)

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.     

Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine epsilon-amino groups during the first 24h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in beta-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the epsilon-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free beta-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no beta-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the beta-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its epsilon-amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101-108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229-237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392], but the lysine-dihydroxyacetophenone compound and the histidine-DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.     

A monoclonal antibody against an adult-specific cuticular protein of Tenebrio molitor (Insecta, Coleoptera)

To study the sequential expression of the epidermal program in the mealworm Tenebrio molitor, monoclonal antibodies were prepared against the water-soluble proteins from preecdysial adult cuticle. Among the 16 clones obtained, one of them (named K2F6) recognized a 20-kDa antigen, found only in adult extracts but not in the larval or pupal ones, as revealed by immunoblot analysis. Our results strongly suggest an epidermal origin for this protein. The monoclonal antibody K2F6 fails to react with water-soluble proteins from fat body and hemolymph taken during the deposition of the 20-kDa antigen. Electron microscopic immunogold localization of this antigen showed that it is secreted, just after epicuticle deposition, in the 30 first-deposited preecdysial lamellae of sternal and elytral cuticles only. The sclerotizing process, which modifies the physicochemical properties of these cuticles, does not prevent the immunoreaction. When the expression of the adult program was inhibited by application of a juvenile hormone analog (ZR 515), the water-soluble proteins from different pupal-adult intermediates were never recognized by the monoclonal antibody K2F6 using immunoblot analysis. These results support the conclusion that this 20-kDa antigen is a protein specific for the sclerotized cuticle of the adult stage.     

Turning behaviour related to optical stimuli in Tenebrio molitor

The walking behaviour of the beetle Tenebrio molitor was studied on a compensating running globe under optical stimuli. The animals show a clear frequency distribution for the duration of continuous walking between abrupt stops. The turning behaviour after a light is switched on or off is correlated with these frequency distributions. The circadian rhythmic changes of activity and of the sensitivity of the eyes influence the turning behaviour. A model describes the phenomenon of ‘sensory storage’ as the result of an integration over the reaction probabilities given by the frequency distributions.     

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.     

A digestive prolyl carboxypeptidase in Tenebrio molitor larvae

Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP.     

我国黄粉虫的营养价值和饲养方法

对我国黄粉虫Tenebriomolitor (L .)的人工饲养、营养价值现状以及当前开发利用研究的热点问题等方面进行了较全面的概括 ,并针对今后的研究工作和规模化生产提出了一些建议。