Tyrosine and catecholamine levels in the hemolymph of tobacco hornworm larvae,Manduca sexta,parasitized by the braconid wasp,Cotesia congregata,and in the developing parasitoids

Parasitism of fifth instar Manduca sexta larvae by the gregarious parasitoid Cotesia congregata prevented normal storage of tyrosine in the hemolymph, whereas total tyrosine levels increased over eight times in the hemolymph of unparasitized larvae by day 4. Tyrosine glucoside, the hemolymph storage form of tyrosine and the precursor for pupal cuticle sclerotizing agents, was found only in trace amounts in parasitized larvae at the time of parasitoid emergence, but had increased to over 6 mM in hemolymph of unparasitized larvae. Concentrations of dopamine and N-β-alanyldopamine (NBAD), precursors for melanization and sclerotization of cuticle, respectively, had approximately doubled in the hemolymph of parasitized larvae by the day of parasitoid emergence, but not in unparasitized larvae. Catecholamine biosynthesis may be transiently stimulated for wound-healing, as black melanic pigmentation appeared around the wasp emergence holes in the host integument. C. congregata larvae accumulate tyrosine, dopamine, and NBAD by the time of emergence and cocoon spinning, either by direct uptake or by synthesis from precursors obtained from the host. NBAD increased in parasitoid larvae close to pupation, suggesting it functions as the main precursor for pupal cuticle tanning. Both dopamine and NBAD increased dramatically in pharate adult wasps just before eclosion and N-acetyldopamine (NADA) appeared for the first time. Dopamine was highest in concentration and total amount, and it can serve both as a precursor for black melanic pigmentation of adult wasp cuticle and for synthesis of NADA and NBAD, the precursors for cuticle sclerotization. Arch. Insect Biochem. Physiol. 38:193–201, 1998. © 1998 Wiley-Liss, Inc.     

Cuticular sclerotization in the beetles Pachynoda epphipiata and Tenebrio molitor

The sclerotization of cuticle in two species of beetles, Pachynoda epphipiata and Tenebrio molitor, has been investigated and compared with the sclerotization in the locust, Schistocerca gregaria. Two types of sclerotization, β-sclerotization and quinone tanning, occur in all three species. The main type is β-sclerotization, i.e. cross-linking of proteins by means of N-acetyldopamine which is connected to the proteins through the β-position of its side chain. β-Sclerotization is completed in P. epphipiata when it leaves its cocoon, whereas in adult locusts and in adult Tenebrio β-sclerotization continues for several weeks. The cuticle of all three species contains an insoluble enzyme which activates the β-position of N-acetyldopamine and is presumably responsible for the formation of the cross-links. Locust cuticle contains also small amounts of another enzyme which activates the aromatic ring of N-acetyldopamine, resulting in the formation of an o-quinone, which may be involved in quinone tanning of the cuticle. At emergence adult Tenebrio cuticle is rich in both enzymes, but the quinone-forming enzyme is inactivated after a few days, whereas the β-enzyme first decreases and later increases in activity, so that the β-enzyme is the dominating activity in the cuticle of mature adult Tenebrio. The quinone-forming enzyme is presumably responsible for the formation of the brown colour of Tenebrio exocuticle.The exocuticle of adult beetles contains 3,4-dihydroxyphenylacetic acid, which, although it is not easily extracted from the cuticle, is not covalently bound to cuticular components. In Tenebrio it appears in the cuticle a few days after the final ecdysis.The amino acid compositions of both larval, pupal, and adult cuticle from P. epphipiata have been determined, and they are compared with the composition of the cuticle of the corresponding stages of Tenebrio.     

Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata

The mechanism of oxidation of two related sclerotizing precursors—N-acetyldopamine and N-acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase-mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N-acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o-dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N-acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N-acetyldopamine-treated cuticle liberated peptides that had covalently bound catechols, while N-acetylnorepinephrine-treated cuticle did not release such peptides. Acid hydrolysis of N-acetyldopamine-treated cuticle, but not N-acetylnorepinephrine-treated cuticle liberated 2-hydroxy-3′,4′-dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N-acetyldopamine to its corresponding quinone methide derivative and N-acetylnorepinephrine to its quinone derivative by the cuticular phen-oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.     

Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

Femur cuticle from fifth instar larvae of the desert locust, Schistocerca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted to the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotization continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned.     

On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or beta-alanine, is connected via its amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the beta-position of the dopamine sidechain. The three ketocatecholic adducts containing alpha-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The beta-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The beta-histidine-dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The beta-histidine-dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.     

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.     

Model sclerotization studies. 3. Cuticular enzyme catalyzed oxidation of peptidyl model tyrosine and dopa derivatives

Incubation of N-acetyltyrosine methyl ester with cuticular enzymes, isolated from the wandering stages of Calliphora sp larvae, resulted in the generation of N-acetyldopa methyl ester when the reaction was carried out in the presence of ascorbate which prevented further oxidation of the o-diphenolic product. Enzymatic oxidation of N-acetyldopa methyl ester ultimately generated dehydro N-acetyldopa methyl ester. The identity of enzymatically produced N-acetyldopa methyl ester and dehydro N-acetyldopa methyl ester has been confirmed by comparison of the ultraviolet and infrared spectral and chromatographic properties with those of authentic samples as well as by nuclear magnetic resonance studies. Since N-acetyldopaquinone methyl ester was also converted to dehydro N-acetyldopa methyl ester and tyrosinase was responsible for the oxidation of N-acetyldopa methyl ester, a scheme for the cuticular phenoloxidase catalyzed conversion of N-acetyltyrosine methyl ester to dehydro N-acetyldopa methyl ester involving the intermediary formation of the quinone and the quinone methide is proposed to account for the observed results. The conversion of N-acetyldopa methyl ester to dehydro derivative remarkably resembles the conversion of the sclerotizing precursor, N-acetyldopamine, to dehydro-N-acetyl-dopamine observed in the insect cuticle. Based on these comparative studies, it is proposed that peptidyl dopa derivatives could also serve as the sclerotizing precursors for the sclerotization of the insect cuticle. © 1995 Wiley-Liss, Inc.     

Involvement of tyrosine residues, N-terminal amino acids, and beta-alanine in insect cuticular sclerotization

During sclerotization of insect cuticle the acyldopamines, N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD), are oxidatively incorporated into the cuticular matrix, thereby hardening and stabilizing the material by forming crosslinks between the proteins in the cuticular matrix and by forming polymers filling the intermolecular spaces in the cuticle. Sclerotized cuticle from the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor, was hydrolyzed in dilute hydrochloric acid, and from the hydrolysates some components presumably degradation products of cuticular crosslinks were isolated. In two of the components, the sidechain of 3,4-dihydroxyacetophenone was linked to the amino groups of glycine and beta-alanine, respectively, and in the third component to the phenolic group of tyrosine. These three compounds, glycino-dihydroxyacetophenone, beta-alanino-dihydroxyacetophenone, and O-tyrosino-dihydroxyacetophenone, as well as the previously reported compound, lysino-dihydroxyacetophenone [Andersen, S.O., Roepstorff, P., 2007. Aspects of cuticular sclerotization in the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor. Insect Biochem. Mol. Biol. 37, 223-234], are suggested to be degradation products of cuticular crosslinks, in which amino acid residues formed linkages to both the alpha- and beta-positions of the sidechain of acyldopamines.     

Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β-sclerotization in the puparia of flesh fly Sarcophaga bullata

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o-diphenolic compounds. While catechol, 3,4-dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 3,4-dihydroxyphenyl glycol, 3,4-dihy-droxymandelic acid, and N-acetyldopamine were oxidized to their side-chain oxygenated products. In addition, the enzyme-catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β-sclerotization. Radioactive trapping experiments failed to support the participation of 1,2-dehydro-N-acetyldopamine as a freely formed intermediate during phenoloxidase-mediated oxidation of N-acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7-³H]N-acetyldopamine and not from either [8-³H] or [ring-³H]N-acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme-catalyzed N-acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2-dehydro-N-acetyldopamine are the reactive intermediate of β-sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.     

Chlorinated tyrosine derivatives in insect cuticle

A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine derivatives were present in all analyzed regions in mature adult locusts, the highest concentrations were found in the sclerotized cuticle of femur and tibia, but significant amounts were also present in the unsclerotized arthrodial membranes. Small amounts of the two amino acids were obtained from pharate, not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed during sample hydrolysis. Mono- and dichlorotyrosine are also present in cuticular samples from other insect species, such as the beetle, Tenebrio molitor, the moth Hyalophora cecropia, the cockroach Blaberus craniifer, and the bug Rhodnius prolixus, but not in the sclerotized puparial cuticle of the blowfly, Calliphora vicina, or in sclerotized ootheca from the cockroach, Periplaneta americana. Cuticular sclerotization and formation of chlorotyrosines occur simultaneously in locust legs; sclerotized cuticles tend to have a higher content of chlorotyrosines than unsclerotized cuticles, but it is concluded that the chlorotyrosines are not just a by-product from the sclerotization process.     

Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony

Microinjection of radioactive tyrosine, dopa, and dopamine into mature larvae of Drosophila revealed that the sclerotization pathway is similar but not identical to that in Calliphora: (a) tyrosine is converted to tyrosine-o-phosphate and not to dopa, and (b) the substrate N-acetyldopamine does not accumulate.Larvae of the mutant ebony appear to be similar to the wild type with respect to tyrosine, dopa, and dopamine utilization. About the time of eclosion, however, ebony has twice as much dopamine as normal. Some implications of this are discussed with reference to the mutant phenotype.     

3-O-Hydrosulphato-4-hydroxyphenethylamine (dopamine 3-O-sulphate), a metabolite involved in the sclerotization of insect cuticle

Dopamine 3-O-sulphate (3-O-hydrosulphato-4-hydroxyphenethylamine) was isolated from newly ecdysed cockroaches, Periplaneta americana (L.), and its structure established by chemical and physical techniques and by synthesis. Relatively high concentrations (about 1mumol/g wet. wt.) of dopamine 3-O-sulphate exist in the newly ecdysed insect, and these concentrations decrease sharply as sclerotization of the cuticle proceeds. At least 40% of the radioactivity of (14)C-labelled dopamine 3-O-sulphate injected into newly ecdysed nymphs was recovered in the sclerotized cuticle 7-12 days after the injection. However, less than 1% of the radioactivity of injected dopamine 3-O-[(35)S]sulphate was recovered, and this value was not appreciably different from that for the incorporation of Na(2) (35)SO(4). Apparently, little or none of the sulphate moiety of dopamine 3-O-sulphate is incorporated directly into the cuticle as the intact sulphate ester. These observations are discussed in relation to current concepts of cuticular sclerotization in insects.     

An enzyme from locust cuticle involved in the formation of crosslinks from N-acetyldopamine

Locust cuticle is shown to contain an enzyme activating the β-position in the side chain of N-acetyldopamine. When isolated cuticle is incubated with N-acetyldopamine part of the substrate becomes incorporated into the cuticle and part of it forms soluble reaction products, one of which is identified as a dimer of N-acetyldopamine. In the structure suggested for the dimer both phenolic groups of one molecule of N-acetyldopamine are connected to the β-position of another.     

A Diphenol Oxidase Gene Is Part of a Cluster of Genes Involved in Catecholamine Metabolism and Sclerotization in Drosophila. I. Identification of the Biochemical Defect in Dox-A2 [l(2)37Bf] Mutants

Phenol oxidase, a complex enzyme, plays a major role in the processes of sclerotization and melanization of cuticle in insects. Several loci have been reported to affect levels of phenol oxidase activity, but to date only one structural locus has been identified [Dox-3F (2-53.1+)]. Recently isolated Dox-A2 mutations (2-53.9) are recessive, early larval lethals, which as heterozygotes reduce phenol oxidase activity. A homozygous mutant escaper had weak, completely unpigmented cuticle and unpigmented bristles. Enzyme assays show that Dox-A2 heterozygotes have diphenol oxidase activity reduced to 47-79% of wild type, whereas monophenol oxidase activity, at 94-106% of wild type, is normal. Elevated pool sizes of the diphenol oxidase substrates DOPA, dopamine, and N-acetyldopamine are observed in the mutant, confirming the enzyme assay results. Separation of the three phenol oxidase A component activities on polyacrylamide gels shows that Dox-A2 mutations reduce the activity of only the A2 component. Dox-A2 may identify a structural locus for the A2 component of the diphenol oxidase enzyme system. The Dox-A2 locus is one of 18 loci in the dopa decarboxylase, Df (2L)TW130 region of the second chromosome, at least 14 of which affect the formation, melanization or sclerotization of cuticle in some way. These loci form an apparent cluster of functionally related genes.     

Catechol conjugation with hemolymph proteins and their incorporation into the cuticle of the American cockroach,Periplaneta americana

Newly ecdysed American cockroaches, Periplaneta americana (sixth to last instar) were injected with radioactive dopamine (DA) and hemolymph was collected at 10–60 min post-ecdysis. Size-exclusion chromatography established the presence of at least three proteins that serve as catecholamine carriers. Reinjection of the smaller radiolabeled phenol-bound proteins into newly ecdysed animals results in in vivo aggregation, with the radiolabel bound to large MW proteins (30->200 kDa). In addition, the reinjection of radiolabeled protein of any size resulted in the incorporation of the label into the newly sclerotized cuticle. Hemolymph proteins were synthesized in vivo using [¹⁴C]leucine and subsequently double labeled in vivo with [³H]dopamine. After sclerotization (7 h post-ecdysis) the cuticle was extirpated, hydrolyzed and counted. An identical ratio of ¹⁴C to ³H was found in cuticle extracts as in the double-labeled hemolymph proteins, suggesting that the phenol-bound protein was incorporated in the cuticle unchanged. It appears that the catechol bound to the proteins exists as a β-glucoside.     

The effects of DOPA decarboxylase inhibitors on the permeability and ultrastructure of the larval cuticle of the Australian sheep blowfly, Lucilia cuprina

Larvae of Lucilia cuprina, fed toxic levels of α-methyl DOPA (or other DOPA decarboxylase inhibitors) during the first or second instar, die at the completion of the next moult, soon after exposing their new cuticles. In electron micrographs of newly synthesised cuticle from these treated larvae, the ultrastructure of the lipid-rich outer epicuticle layer appears to be abnormal. This newly formed cuticle of the treated larvae is apparently defective in its role as a water permeability barrier (compared with that of normal larvae), since it permits the free movement of water in both directions. Thus, treated larvae die most probably as a direct result of dehydration. Larvae fed toxic levels of α-methyl DOPA can be rescued from death by simultaneously adding N-acetyldopamine (the cuticular sclerotizing agent) to the food. The rescued larvae are apparently normal in all respects. This suggests that sclerotization is required for the formation of a normal outer epicuticle. Diflubenzuron, which is known to inhibit chitin deposition in the cuticles of a number of different species of insect, also apparently affects chitin deposition in the larval cuticle of L. cuprina. Thus, in electron micrographs of cuticle from larvae fed toxic levels of diflubenzuron the ultrastructure of the chitin-containing endocuticle layer appears to be abnormal.     

Regional differences in degree of resilin cross-linking in the desert locust, Schistocerca gregaria

Various cuticular regions from the desert locust, Schistocerca gregaria, were quantitatively analyzed for two cross-linking amino acids, dityrosine and trityrosine, characteristic constituents of the rubberlike cuticular protein, resilin. These amino acids were found in all regions of cuticle investigated, but in widely varying amounts. In fully mature adult locusts the largest amounts of di- and trityrosine were obtained from the prealar arms and wing-hinges, structures possessing long-range elasticity and being involved in energy storage in the flight system. In structures where deformations tend to occur more slowly, such as the clypeo-labral springs and tracheae, di- and trityrosine are less abundant. In sclerotized cuticle from femur and tibia, as well as in cornea and in the highly stretchable intersegmental membranes of mature females, they are only found in trace amounts and are probably unrelated to elasticity. The trityrosine-to-dityrosine ratio in the various cuticular regions vary from nearly equal amounts of the two amino acids to about ten times more dityrosine than trityrosine, indicating that the regions differ in degree of cross-linking; the tracheal wall is the material with the highest trityrosine-to-dityrosine ratio. In some cuticular regions the ratio increases during maturation from newly moulted (teneral) adults to reproductively active locusts; the most pronounced increase was observed for the wing-hinges, and only a small increase was observed for the abdominal tergal plates. In most cuticular regions in fifth instar locust nymphs the contents of di- and trityrosine corresponded to the contents measured for the adult cuticular regions, but only trace amounts of the two amino acids were obtained from the region of the nymphal wing base which corresponds to the wing-hinge containing cuticular region in adult locusts.     

Enzymic activities involved in the oothecal sclerotization of the praying mantid,Tenodera aridifolia sinensis saussure

The enzyme(s) responsible for the sclerotization of mantid ootheca is secreted by the left colleterial gland. From an extract of the glands of Tenodera aridifolia sinensis, two soluble enzyme fractions of different activities were obtained. One fraction acted on N-acetyldopamine (NADA), a precursor of a representative sclerotizing agent, and produced NADA-quinone. The other did not act on NADA itself but converted the quinone to a highly reactive intermediate, such as quinone methide, which was able to react nonenzymically with nucleophilic compounds. Other insoluble enzyme preparations obtained from the silk and pupal cuticle of the Japanese giant silk moth, Dictyoploca japonica, also had these two activities.