Characterization and cDNA cloning of three major proteins from pharate pupal cuticle of Manduca sexta.

Three proteins, MsCP20, MsCP27 and MsCP36, that are secreted in greatest quantity into the pharate pupal cuticle of Manduca sexta ( Hopkins et al., 2000) were purified and their amino acid sequences determined by mass spectrometry and Edman degradation. Although these proteins become sclerotized and insoluble in the pupal exoskeleton, their sequences contain features characteristic for proteins occurring in less sclerotized pliable cuticles, such as arthrodial membranes and soft larval cuticles. These proteins carry a secondary modification attached to a threonine residue, presumably an O-linked sugar moiety. cDNA clones of the genes for MsCP20, MsCP27 and MsCP36 were constructed from pharate pupal integument RNA. Close agreement was found between the amino acid sequences determined by Edman degradation and sequences deduced from the cDNA clones. The molecular masses determined by protein sequencing for MsCP20, MsCP27, and MsCP36 were 17713, 17448, and 29582 Da, respectively, in close agreement with the masses deduced from the corresponding cDNA clones (17711, 17410, and 29638 Da). Temporal expression analysis indicates that MsCP20 and MsCP36 transcripts are present at low levels early in the fifth larval stadium, followed by a large increase in abundance prior to pupal ecdysis. MsCP27 was not detected during development of the fifth larval instar, but its transcript, like those of MsCP20 and MsCP36, increased to a peak level just before pupal ecdysis. Only the MsCP36 transcript was detected in adults. These results support the hypothesis that these proteins are synthesized by the epidermis and are subsequently deposited into the cuticle during the larval-pupal transformation of M. sexta where they become sclerotized in the formation of pupal exocuticle.

Lepidopteran peritrophic membranes and effects of dietary wheat germ agglutinin on their formation and structure.

Peritrophic membrane (PM) structure and the effects of dietary wheat germ agglutinin (WGA) on PM formation were studied in larvae of the European corn borer (ECB), Ostrinia nubilalis, and the tobacco hornworm (THW), Manduca sexta. Growth of ECB was strongly inhibited by low amounts of WGA in the diet (0.05%), whereas THW was not affected by amounts of up to 2%. In ECB larvae, chitin microfibrils were secreted to form an orthogonal network within the apical region of the anterior midgut microvilli. The network then moved to the tips of the microvilli where proteinacious matrix was added prior to delamination of a single PM into the lumen to enclose the food bolus. Multiple PMs rapidly appeared as the food moved posteriorly and some of these became greatly thickened in the middle and posterior regions of the midgut. WGA in the diet caused hypersecretion of unorganized PM in the anterior midgut lumen, disintegration of microvilli, and cessation of feeding. It was also shown to bind to both the chitinous network and to several PM proteins, perhaps causing voids in the PM and sparse matrix material. This allowed the passage of food particles through a defective PM into the ectoperitrophic space and penetration into the microvillar brush border. Stimulation of PM secretion and cessation of feeding may have been a response to damage to the brush border. Unlike ECB, the chitinous network of THW is a randomly organized felt-like structure embedded in a proteinaceous matrix. This PM is secreted as a thin multilayered structure in the anterior region of the midgut, but multiple and thickened PMs occur in the middle and posterior lumens of the midgut. THW tolerated high amounts of WGA in its diet with no disruption of PM formation or inhibition of growth. WGA did accumulate as large masses embedded in the PM, but caused no voids that would allow the penetration of food particles and subsequent damage to the brush border. Therefore, differences in PM formation and structure between ECB and THW appeared to affect how WGA interacts with chitinous and proteinaceous components of the PM and subsequent effects on larval feeding and growth.

Pupal cuticle proteins of Manduca sexta: characterization and profiles during sclerotization.

Proteins in pupal abdominal cuticle of the tobacco hornworm, Manduca sexta, were characterized during the pre-ecdysial and post-ecdysial periods of sclerotization and endocuticle formation. Protein extractability decreased dramatically as the cuticle became sclerotized through 6 h post-ecdysis, but increased rapidly from 9 to 48 h as endocuticular layers were secreted. Nearly 100 proteins that were extracted from pre-ecdysial cuticle became largely insoluble during sclerotization. Three major proteins in this group destined to become exocuticle had apparent molecular masses (Mapp) of 20, 27 and 36 kDa, and were designated MS-PCP20, MS-PCP27, and MS-PCP36. Amino acid analysis revealed glycine to predominate in all three proteins, and alanine, aspartate, glutamate, proline and serine were also relatively abundant. Histidine residues, which provide sites for adduct and cross-link formation with quinone metabolites of N-beta-alanyldopamine during sclerotization of pupal cuticle, ranged from 2 to 3 mol %. N-Terminal amino acid analysis of MSPC-20 and MSPC-36 also revealed some sequence similarities indicating they may be related. An almost entirely new group of proteins appeared by 9 h as endocuticule secretion began, and these increased in abundance through 48 h post-ecdysis. Two of these were major proteins with Mapps of 33 and 34 kDa, and they also had close similarities in their N-terminal amino acid sequences. This study showed that the large number of proteins secreted into the presumptive exocuticle of the pupa before ecdysis are involved in sclerotization reactions and as a consequence become largely insoluble. The epidermis then switches to the secretion of an entirely new group of proteins that are involved in formation of the endocuticle.

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle.

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.

The effects of perinatal hypoxic risk on developmental outcome in early and middle childhood: a twin study.

The goal of this study of 66 twins was to determine whether motor and cognitive functions assessed in early and middle childhood are vulnerable to perinatal hypoxic risk. In an earlier study of 76 infant and toddler twins (S. Raz, F. Shah, & C. Sander, 1996), the authors found that intrapair discrepancy on the Mental Developmental Index, but not on the Psychomotor Developmental Index, of the Bayley Scales of Infant Development was associated with discordance for perinatal hypoxic risk. The twins at lower risk outperformed their higher risk co-twins. In the present study the authors sought to establish in a new sample of preschool and school-age twins whether gaps in performance persist into early and middle childhood. Although the disparity in hypoxic risk between the co-twins was typically moderate, significant intrapair differences were observed on the measure of motor performance. Among the motor abilities examined, skills involving visually guided ballistic arm movements appeared to be the most vulnerable to perinatal risk.

Electrochemical oxidation of N-acyldopamines and regioselective reactions of their quinones with N-acetylcysteine and thiourea.

The metabolism of catechols often involves their oxidation to quinones and subsequent nucleophilic addition reactions with sulfur-containing compounds. Adducts formed during these reactions may play important roles in many biological systems. We have studied the electrochemical oxidation of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD) in the presence of two sulfur-centered nucleophiles, N-acetylcysteine (NACySH) and thiourea (TU), and have characterized the adducts and reaction pathways. NADA and NBAD react similarly, but their adducts with NACySH and TU were formed regioselectively. NACySH yields mainly 5-adducts and TU only 6-adducts. The NACySH adducts are oxidized more easily than the parent N-acyldopamine, and their oxidations are chemically reversible. However, the TU adducts are more difficult to oxidize, and their oxidation products undergo further chemical reactions. An intramolecular base catalysis mechanism for adduct formation with NACySH is proposed, which facilitates removal of the proton from the sulfhydryl group of NACySH and directs formation of the 5-adduct via a 1,6-Michael addition reaction. The absence of a proton on the thioureylene sulfur atom leads to formation of the 6-thioureylene adduct via a 1,4-Michael addition reaction of TU. This mechanism is consistent with the formation of other sulfur-centered adducts of catechols previously reported in the literature.

Effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in European corn borer (Ostrinia nubilalis) larvae.

European corn borer (ECB; Ostrinia nubilalis (Hubner)) larvae (third instar) fed 0.05% w/w wheat germ agglutinin (WGA) in their diet for 72 h showed very little increase in body weight, whereas weight of control larvae increased nearly fourfold. Light and transmission electron microscopy studies showed that the morphology of the peritrophic membrane (PM) changed within 24 h after ECB larvae fed on the WGA diet. Whereas the PM in the anterior region of the midgut was a thin membranous structure in control larvae, the WGA-fed larvae secreted a multiple-layered and unorganized PM that contained embedded food particles, bacteria, and pieces of disintegrated microvilli. Gold-labeled WGA was localized specifically in the PM and microvilli. The PM of WGA-fed larvae was inundated with dark-staining amorphous structures that, when incubated with anti-WGA, showed heavy WGA localization. The antibody label indicated that most of the ingested WGA was found in the PM, with lesser amounts on the microvillar surface and the least amount within the epithelium. After 72 h, the middle portion of the mesenteron revealed a thin, compact PM in the control larvae, whereas the PM of the WGA-fed larvae was multilayered and discontinuous, which allowed plant cell-wall fragments to penetrate into the microvilli of the epithelium. Scanning electron microscopy of PMs from fifth instar ECB larvae fed the WGA diet revealed a breakdown in the chitinous meshwork by 48 h after initiation of feeding. The endo-PM surface from control larvae was smooth and intact, whereas the PM of WGA-fed larvae showed disintegration of the meshwork and a reduced proteinaceous matrix. This allowed bacteria and food particles to penetrate through the PM into the ectoperitrophic space and directly contact the microvilli. Therefore, WGA, a protein inhibitor of larval growth, interferes with the formation and integrity of the PM, which exposes the brush border to ingested material. This, in turn, appears to stimulate secretion of additional PM layers, the concomitant disintegration of the microvilli, and cessation of feeding.

Peritrophic membrane structure and secretion in European corn borer larvae (Ostrinia nubilalis).

Peritrophic membrane (PM) secretion and formation occur primarily in the anterior region of the mesenteron in the European corn borer (Ostrinia nubilalis) as determined by light and electron microscopy. Nascent PM first became visible as fibrous linear chitin-containing structures stained with gold-labeled wheat germ agglutinin between and at the tips of the microvilli. No formed PM was visible at the foregut-midgut junction, but a thin single PM appeared first in the lumen between the stomodeal valves and the midgut epithelium. Just posterior to the stomodeal valves, multiple PMs were observed that became progressively thicker and more numerous in the mid and posterior regions of the mesenteron. The PM consists of an orthogonal chitin meshwork with openings slightly larger than the diameters of the microvilli. As it delaminates from the microvilli, the meshwork becomes embedded in proteinaceous matrix that greatly reduces the pore size of the PM.

Catecholamine-containing proteins from the pharate pupal cuticle of the tobacco hornworm, Manduca sexta.

Three catecholamine-containing proteins from tanning, pharate pupal, abdominal cuticle of the tobacco hornworm, Manduca sexta, have been purified to apparent homogeneity and characterized. These proteins have apparent molecular masses of 32, 41 and 48 kDa and were shown by liquid chromatography-electrochemical analysis, after heating in 1 M acetic acid at 110 degrees C, to release nu-beta-alanylnorepinephrine (NBANE). NBANE is a hydrolysis product of N-beta-alanyldopamine (NBAD) that is bonded covalently at the beta-side-chain carbon to amino acid residues of cuticular proteins. Amino acid compositional analysis revealed that MS-PCP32 (32 KDa) has a high content of alanine (25.6%), valine (13.1%), proline (10.8%) and glycine (10%), a low level of phenylalanine, and no detectable tyrosine or methionine. In contrast, MS-PCP41 (41 kDa) had a much higher content of glycine (31.2%) and substantial levels of serine (9.2%), proline (10.2%) and glutamate/glutamine (10.6%), whereas tyrosine and phenylalanine were not detected. Two of the three purified proteins showed apparent similarities to each other in nu-terminal amino acid sequences, and to several other known insect cuticular proteins. Proteins MS-PCP41 and MS-PCP48 had the characteristic GGX triplet repeat, which is found in a variety of cuticular proteins and may be important for protein folding appropriate for cuticular functions. Therefore, a diversity of cuticular proteins with different amino acid sequences and properties apparently are secreted into the presumptive pupal exocuticle. These then can form adducts and possible cross-links with NBAD through its quinonoid intermediates during cuticular sclerotization.

Characterization of two high molecular weight catechol-containing glycoproteins from pharate pupal cuticle of the tobacco hornworm, Manduca sexta.

Two high molecular weight cuticular proteins (MSCP120 and MSCP246) were extracted in acidic guanidine hydrochloride solution from tanning abdominal cuticle of Manduca sexta pharate pupae and purified by size exclusion high performance liquid chromatography. The apparent molecular weights were ca. 120 and 246 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both proteins contained high levels of glutamate/glutamine, glycine, serine, alanine and aspartate/asparagine. MSCP120 was enriched in histidine relative to MSCP246, but the reverse was true for valine and proline. Small quantities of beta-alanine and 3,4-dihydroxyphenylalanine (DOPA), as well as other catechols and carbohydrates, also were detected in the hydrolysates. The proteins became radiolabeled when [1-14C]-beta-alanine was injected into pharate pupae, presumably by the formation of adducts with N-beta-alanyldopamine metabolites during sclerotization. Mild acid hydrolysis released N-beta-alanylnorepinephrine and 3,4-dihydroxyphenylketoethanol from both proteins. Strong acid hydrolysis yielded predominantly 3,4-dihydroxyphenylketoethylamine (arterenone), but also DOPA and dopamine. The N-terminal amino acid sequences of the two cuticular proteins were dissimilar, and that of MSCP246 was more hydrophobic than MSCP120. Both of these proteins were glycosylated with glucose, N-acetylglucosamine and traces of N-acetylgalactosamine, and MSCP246 also contained galactose. These structural glycoproteins, which occur in cuticle undergoing sclerotization, apparently react post-translationally with quinonoid tanning agents to yield catecholamine-protein adducts. Small amounts of peptidyl DOPA probably are formed by hydroxylation of tyrosyl residues. Results from this study are consistent with the hypothesis that these catechol-containing glycoproteins participate in cross-linking reactions in M. sexta pupal cuticle during sclerotization.

1H and 13C NMR of 3-O and 4-O conjugates of dopamine and other catecholamines.

Dopamine and its conjugates are widely distributed among biological species and are utilized for a variety of functions. Insects metabolize dopamine for cuticle melanization and sclerotization. Among the most abundant dopamines found in the larval and pupal development stages of Manduca sexta, the tobacco hornworm, are N-acetyldopamine and N-beta-alanyldopamine. In addition, glycosylated derivatives of these dopamines are found mainly in the hemolymph just prior to cuticulogenesis. The 1H and 13C NMR resonances of dopamine, its 3-O-methyl, 4-O-methyl, N-acetyl, and N-beta-alanyl derivatives, norepinephrine, 4-O-(beta-D-glucuronopyranosyl)dopamine, and the glycosylated products of N-beta-alanyldopamine and dopamine have largely been assigned. Assignments were based on one- and two-dimensional NMR analyses of the above compounds combined with that of specifically enriched [C7-13C]dopamine. 1H NMR showed that the major glycosylated natural product isolated from M. sexta pupal hemolymph was a 3-O-glycosyl derivative of N-beta-alanyldopamine. 13C NMR confirmed that the carbohydrate was D-glucose probably in a beta-linkage. 1H NMR of the aromatic ring protons provided the most definitive method to distinguish 3-O- from 4-O-derivatives of dopamine. In addition, the 3-O-glucosyl conjugate of N-beta-alanyldopamine had unique chemical shifts and coupling patterns compared to those of 4-O-(beta-D-glucuronosyl)- and 3-O-(beta-D-glucopyranosyl)dopamine.

Comparison of black coral skeleton and insect cuticle by a combination of carbon-13 NMR and chemical analyses.

Cross-polarization, magic-angle spinning 13C NMR spectra of skeletal components of individual colonies of the New Zealand black coral, Antipathes fiordensis, have a marked similarity to spectra of the sclerotized exoskeleton of the adult tobacco hornworm, Manduca sexta. NMR analysis estimates the organic content of the load-bearing skeletal base of A. fiordensis as 70% protein, 10% chitin, 15% diphenol, and 5% lipid by weight, and that of M. Sexta moth cuticle as 60% protein, 20% chitin, 15% diphenol, and 5% lipid. The younger pinnules or tips of A. fiordensis are less than 3% diphenol by weight. The only diphenols extracted from coral skeleton by hydrochloric acid are 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) and 3,4-dihydroxybenzaldehyde (DOBAL), while the predominant diphenols in acid extracts of insect cuticles are N-acyldopamines. More DOPA is found in the base than in the tips of A. fiordensis and it appears to be a peptidyl component of coral skeletal protein. The oxidation of DOPA and DOBAL to quinones may provide mechanical stabilization of the coral skeleton by cross-linking of structural proteins to other proteins or to chitin.

Oxidative decarboxylation of 3,4-dihydroxymandelic acid to 3,4-dihydroxybenzaldehyde: electrochemical and HPLC analysis of the reaction mechanism.

Cyclic voltammetric and chronoamperometric data are consistent with a process in which 3,4-dihydroxymandelic acid (DOMA) is oxidized initially in a two-electron step to its corresponding o-benzoquinone. This species is unstable and undergoes the rate-determining loss of CO2 (k = 1.6 s-1 at pH 6 and 25 degrees C) to give an unobserved p-benzoquinone methide intermediate that rapidly isomerizes to 3,4-dihydroxybenzaldehyde (DOBAL), DOBAL is also electroactive at the applied potential and is oxidized in a two-electron step to 4-formyl-1,2-benzoquinone. Subsequent reactions of 4-formyl-1,2-benzoquinone include the oxidation of unreacted DOMA and the hydration of its aldehyde functional group. Oxidation of DOMA directly to its p-benzoquinone methide apparently does not occur. Derivatives of mandelic acid (e.g., 4-hydroxymandelic acid) that are expected to give only their corresponding p-benzoquinone methides upon oxidation afford redox behavior that differs distinctly from that for DOMA.

Catecholamines and related o-diphenols in cockroach hemolymph and cuticle during sclerotization and melanization: comparative studies on the order Dictyoptera.

Catecholamines and related o-diphenols extracted from the cuticle and hemolymph of adult cockroaches during sclerotization and pigmentation of the cuticle were analyzed by reverse phase HPLC with electrochemical detection. At ecdysis, dopamine (DA) o-conjugates predominated in the hemolymph of Periplaneta americana, P. australasiae, P. fuliginosa, P. brunnea, and Blatta orientalis (Blattidae); Blattella germanica (Blattellidae); and Gromphadorhina portentosa and Blaberus craniifer (Blaberidae). N-Acetyldopamine (NADA) conjugates were second in abundance in these species, but were major in the hemolymph of the other blaberoid species, Leucophaea maderae and Nauphoeta cinerea. After ecdysis NADA became the major hemolymph catecholamine in all species as DA decreased rapidly. N-beta-Alanyldopamine (NBAD) concentrations in the hemolymph remained low in all species, although NBAD and its metabolite, N-beta-alanylnorepinephrine (NBANE), were generally the major catecholamines in tanning cuticle. Catechol (1,2-dihydroxybenzene) occurred mainly as a conjugate(s) at high levels in the hemolymph of nymphs and adults of all blattid species. Only trace amounts were detected in B. germanica and Cryptocercus punctulatus (Cryptocercidae), and none was found in any of the blaberoid species. High concentrations of NBANE and NBAD accumulated in tanning cuticle of B. germanica, G. portentosa, and all blattid species, whereas NADA and DA predominated in cuticle from the other blaberoid species, particularly L. maderae and N. cinerea. However, cockroaches as a group appear to utilize both the N-acetyl and N-beta-alanyl catecholamines for stabilization of the exoskeleton. The Blattidae differed most from the other families in having considerably higher concentrations of catecholamines in hemolymph and cuticle, as well as the large amounts of catechol conjugates in the hemolymph.

Aromatic cross-links in insect cuticle: detection by solid-state 13C and 15N NMR.

Cross-polarization magic-angle-spinning nuclear magnetic resonance spectroscopy has been used to determine insect cuticle composition and cross-link structure during sclerotization or tanning. Unsclerotized cuticle from newly ecdysed pupae of the tobacco hornworm, Manduca sexta L., had a high protein content with lesser amounts of lipid and chitin. Concentrations of chitin, protein, and catechol increased substantially as dehydration and sclerotization progressed. Analysis of intact cuticle specifically labeled with carbon-13 and nitrogen-15 revealed direct covalent linkages between ring nitrogens of protein histidyl residues and ring carbons derived from the catecholamine dopamine. This carbon-nitrogen adduct was present in chitin isolated from cuticle by alkaline extraction and is probably bound covalently to chitin. These data support the hypothesis that the stiffening of insect cuticle during sclerotization results primarily from the deposition of protein and chitin polymers and their crosslinking by quinonoid derivatives of catecholamines.

Olfactory responses of a specialist and a generalist grasshopper to volatiles ofArtemisia ludoviciana nutt. (Asteraceae).

Hypochlora alba is a specialist grasshopper that lives and feeds almost exclusively onArtemisia ludoviciana, a plant that produces large amounts of allelochemics including a variety of monoterpenes. This plant is not a host for generalist grasshoppers such asMelanoplus sanguinipes. The role of olfaction in the grasshopper-plant relationship was investigated by comparing electroantennograms (EAGs) of males and females of both species generated by solvent-extracted volatiles from plant leaves and by major individual components. Volatiles ofA. ludoviciana were identified by gas chromatography-mass spectrometry. The major components were 1,8-cineole, camphor, camphene, and borneol, while minor identified compounds were α- and ß-pinene, α-thujene, myrcene,p-cymene, Artemisia ketone, α-thujone, and bornyl acetate. The EAGs (mV) ofH. alba males to a range of concentrations of individual volatiles or the total plant extract were nearly double those of conspecific females or both sexes ofM. sanguinipes. However, both sexes ofM. sanguinipes were more sensitive than either sex ofH, alba to geraniol, a monoterpene that commonly occurs in many plant species but is absent or is present in only trace amounts inA. ludoviciana. The increased sensitivity ofH. alba males to the odor components of their host plant appears to be related to the greater number of certain olfactory chemoreceptors on male versus female antennae. The significance of this phenomenon is discussed.

Antarctic mesopelagic micronekton: evidence from seabirds that pack ice affects community structure.

Through a multidisciplinary project (AMERIEZ), with an unusual complement of components, previously unknown temporal and spatial dimensions to the structure of Antarctic epipelagic and mesopelagic communities were revealed. In late spring, an abundance of crustacean species thought to occur only below 300 meters was detected in ice-covered surface waters. Evident in ice-free waters were the expected occurrence patterns of these normally nonmigratory mesopelagic organisms. Where the pack was consolidated and little light penetrated to depth, primary and secondary production was confined to ice floes, and the physical environment immediately beneath the ice was reminiscent of a mesopelagic one. This suite of characteristics possibly explains why the crustaceans resided at the surface.

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone.

When Manduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both alpha-MDH and alpha-fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 microgram/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.

N-beta-Alanyldopamine: Major Role in Insect Cuticle Tanning.

N-beta-Alanyldopamine is the major tyrosine metabolite in the hemolymph and cuticle during pupal tanning in the tobacco hornworm, Manduca sexta L. Its concentration in hemolymph increases over 800-fold above larval levels by the start of tanning and decreases as the pupal cuticle darkens and hardens. It is a major catechol in species representing several insect orders and is the preferred substrate for pupal cuticular o-diphenol oxidase. In insects, N-beta-alanyldopamine appears to be the main precursor for tanning chemicals at certain developmental stages.

Olfactory responses of adultTribolium castaneum (Herbst), to volatiles of wheat and millet kernels, milled fractions, and extracts.

Olfactory responses of 72- to 96-hr-old, 24-hr starved adult male, female, or mixed-sex groups of the red flour beetle,Tribolium castaneum (Herbst), to volatiles from wheat and millet kernels, certain milled fractions, and solvent extracts were recorded by using a lightsensitive apparatus. Wheat-germ volatiles from intact germ or solvent extracts were generally more attractive than volatiles of wheat endosperm or wheat bran. Volatiles of whole wheat kernels were the least attractive among test materials of wheat origin, whereas there were no significant differences among whole wheat flour, germ, and endosperm. Wheat-germ extracts, however, were more attractive than were extracts of the other fractions. Whole millet flour or fermented millet flour volatiles were more attractive than those from whole millet kernels or millet starch. Beetles reached maximum responsiveness to grain volatiles by 72-96 hr after adult ecdysis. Groups of virgin female beetles generally were more responsive than male or mixed-sex groups to volatiles of substances tested.