A post-ecdysial plasticization of the abdominal cuticle in Rhodnius

The ecdysis and emergence of fifth instar larvae of Rhodnius prolixus have been closely observed. At the time of ecdysis the cuticle of the head, legs, and wingpads is soft and readily deformed. it does not become sufficiently rigid for normal use until about 90 min later. The cuticle of the abdomen is however hard and inextensible at the time of ecdysis. From about 60 min onward this cuticle undergoes a plasticization; it is maximally extensible at about 180 min, thereafter becoming inextensible again. Unlike the plasticization of the abdominal cuticle which occurs after feeding, this post-ecdysial plasticization is not under direct nervous control. Although it seems that there is some temporal link with the darkening of the cuticle, it is considered unlikely that plasticization is a direct consequence of the tanning process. The significance of this post-ecdysial plasticization is not obvious.     

High Laccase2 expression is likely involved in the formation of specific cuticular structures during soldier differentiation of the termite Reticulitermes speratus

Termite soldiers are morphologically specialized for colony defense. Analysis of the mechanisms of soldier differentiation is important for understanding the establishment of termite societies. Soldiers differentiate from workers through a presoldier stage and have well-sclerotized and pigmented cuticles. These characteristics are important for nest defense and are likely to be caused by soldier-specific mechanisms of cuticular tanning. The molecular mechanisms leading to cuticular tanning have not been elucidated. Laccase2 (Lac2) plays important roles in this process in insects, and we hypothesized that Lac2 expression may be involved in soldier-specific cuticular tanning. We observed inner and outer head cuticle changes and compared the Lac2 expression patterns among three molts (worker–worker, worker–presoldier and presoldier–soldier) in the termite Reticulitermes speratus. Quantitative analyses of head cuticle colors showed that the color properties changed more conspicuously in presoldier–soldier molts than in the other two molts. Histological observations showed that the exocuticles of soldier heads were substantially thicker than those of worker and presoldier heads, underwent tanning before or just after ecdysis, and were pigmented at earlier time points than other molts. Finally, markedly higher Lac2 expression levels were observed just before and after ecdysis only in the presoldier–soldier molt. These results suggest that specific cuticular formation occurs in the exocuticles during soldier differentiation, and that the high level of Lac2 expression during the presoldier–soldier molt is related to soldier-specific cuticular tanning. We speculate that evolution of the regulatory mechanisms of Lac2 expression were important for the acquisition of soldier-specific cuticles.     

Effects of catecholamines and β-alanine on tanning of pupal cuticle of Manduca sexta in vitro

When epidermis from wandering stage tobacco hornworm (Manduca sexta) larvae was exposed to 5 μg/ml 20-hydroxyecdysone for 3 days, then exposed to hormone-free Grace's medium, the newly formed pupal cuticle tanned slowly up to 35% of its area by day 12. The addition of 1.3 mM dopamine on either day 4 or 5 slightly increased the area tanned and addition of β-alanine (to 11.2 mM) on days 3–5 enhanced tanning 2–2.5-fold by day 8. Later addition had no effect. When pharate pupal cuticle about 24 h before ecdysis was explanted to Grace's medium, little tanning occurred in 24 h unless dopa or dopamine or their derivatives were added; β-alanine up to 4.4 mM had no effect. Partial tanning occurred in 10 mM dopa or dopamine. More effective were N-β-alanylnorepinephrine and N-β-alanyldopamine which produced nearly maximal tanning at 1 and 5 mM respectively. Up to 10 mM N-β-acetylnorepinephrine had little effect. Thus, dopamine and β-alanine are important to cuticular tanning in vitro and apparently need to be incorporated into the exocuticle during its synthesis. Maximal tanning of this exocuticle then requires further incorporation of β-alanyl conjugates.     

Egg chorion tanning in Aedes aegypti mosquito

The biochemical pathway of egg chorion tanning in the mosquito, Aedes aegypti, is described and compared with chorion protein crosslinking in Drosophila and silkmoths and the biochemical pathways of cuticular tanning in insects. Phenol oxidase, dopa decarboxylase and tyrosine are critical components involved in egg chorion tanning in A. aegypti. Tanning of the mosquito egg chorion is initiated following activation of phenol oxidase, which then catalyzes the hydroxylation of tyrosine to dopa and further oxidizes dopa and dopamine to their respective o-quinones. Because intramolecular cyclization is much slower in dopaminequinone than dopaquinone, the chance to react with external nucleophiles to participate in protein crosslinking reactions also is much greater in dopaminequinone than dopaquinone. This might partly explain the necessity for the involvement of dopa decarboxylase in mosquito chorion tanning. Intramolecular cyclization of dopaquinone and dopaminequinone to form dopachrome and dopaminechrome, respectively, the structural rearrangement of these aminochromes to produce 5,6-dihydroxyindole, and the subsequent oxidation of 5,6-dihydroxyindole by phenol oxidase also lead to melanin formation during egg chorion tanning.     

Properties of pullulan-based blend films as affected by alginate content and relative humidity

Pullulan-sodium alginate blend films were prepared and characterized as a function of water activity (a_w). At low a_w, the incorporation of alginate into pullulan film increased the tensile strength and elastic modulus, but decreased the elongation at break of the composite films; the opposite trends were observed at elevated a_w. Above 0.43 a_w, water exerted a typical plasticization effect upon the biopolymer blends. As a_w increased from 0.23 to 0.43, an anti-plasticization effect was observed as tensile strength and elastic modulus increased. The glass transition temperature of all samples decreased substantially as aw increased from 0.23 to 0.84 due to the plasticization effect of water. Within this a_w range, one transition temperature was observed for all film specimens. The stretching vibration band of O-H was investigated using attenuated total reflection Fourier transform infrared spectroscopy to identify the various species of water interacting with the polysaccharide films.     

Hydration and tanning in insect cuticle

The water content of larval and puparial cuticle of Calliphora vomitoria has been measured under differing conditions using nuclear magnetic resonance, differential scanning calorimetry and simple gravimetry. On average, 2.5 molecules of water are associated with each amino acid side chain. This water is not displaced by tanning, even though tanning reduces the overall content of freezable water, suggesting that tanning agents do not interact with polar groups on the protein but increase overall hydrophobicity. This refutes normally accepted concepts of tanning by covalent cross linking. Additionally, covalent cross linking cannot account for the reduction in swellability of the cuticle on tanning.     

Metallic gold and silver colours in some insect cuticles

Broad bandwidth physical gold and silver cuticular interference reflectors were studied with a microspectroscope. Plusiotis optima adults reflect wavelengths throughout the human visible spectrum, appearing silver because of their high reflectivity. In P. gloriosa, the silver stripes are broad bandwidth interference reflectors whereas the green stripes reflect a narrow bandwidth. Pupae of Heliconius erato have regions which are silver and other parts where physical silver is combined with chemical tanning colouration to produce a gold colour. Developmental changes of silver in Antheraea pernyi larvae, of gold in adult Aspidomorpha tecta, and of silver and gold in pupal Heliconius erato are described. Reversible colour changes in living A. tecta adults were mimicked by placing pieces of cuticle in buffer solutions of different pH. During starvation the endocuticle which produces the physical colour is resorbed and the brightness reduces. The diversity of golds and silvers is discussed with reference to location of layers responsible, tanning, pigments, extracellular pH control, cuticular hydration, and possible defence mechanisms.     

The chemical nature of the egg-shell of helminths. I. Absence of quinone tanning in the egg-shell of the liver fluke, Fasciola hepatica

Ramalingam K. 1973. The chemical nature of the egg-shell of helminths—I. Absence of quinone tanning in the egg-shell of the liver fluke, Fasciola hepatica. International Journal for Parasitology, 3: 67–75. The mode of stabilization of the egg-shell protein of Fasciola hepatica has been studied. It was observed that quinone tanning is absent in the egg-shell. Unlike quinone-tanned proteins, egg-shell proteins show auto-fluorescence. Investigation on the fluorescent compounds present in the egg-shell protein shows the presence of di-tyrosine. In addition to the presence of di-tyrosine, the egg-shell protein is stabilized by —S—S—bonding. The resistant properties of the egg-shell of F. hepatica are attributed to the presence of the above types of cross-links.     

A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)

The levels of ecdysteroids in Sarcophaga bullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa, the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle.Assays for the cuticle tanning hormone, bursicon, in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed.     

Mechanical properties of the beetle elytron, a biological composite material

We determined the relationship between composition and mechanical properties of elytra (modified forewings that are composed primarily of highly sclerotized dorsal and less sclerotized ventral cuticles) from the beetles Tribolium castaneum (red flour beetle) and Tenebrio molitor (yellow mealworm). Elytra of both species have similar mechanical properties at comparable stages of maturation (tanning). Shortly after adult eclosion, the elytron of Tenebrio is ductile and soft with a Young's modulus (E) of 44 ± 8 MPa, but it becomes brittle and stiff with an E of 2400 ± 1100 MPa when fully tanned. With increasing tanning, dynamic elastic moduli (E') increase nearly 20-fold, whereas the frequency dependence of E' diminishes. These results support the hypothesis that cuticle tanning involves cross-linking of components, while drying to minimize plasticization has a lesser impact on cuticular stiffening and frequency dependence. Suppression of the tanning enzymes laccase-2 (TcLac2) or aspartate 1-decarboxylase (TcADC) in Tribolium altered mechanical characteristics consistent with hypotheses that (1) ADC suppression favors formation of melanic pigment with a decrease in protein cross-linking and (2) Lac2 suppression reduces both cuticular pigmentation and protein cross-linking.     

Role of beta-alanine in cuticular tanning,sclerotization, and temperature regulation in Drosophila melanogaster

Injection of 20 nl of 1.0 M beta-alanine, about the minimal amount needed to produce wild-type tanned phenomenocopies from newly eclosed mutant black Drosophila melanogaster, increases stiffness and puncture-resistance of the wing cuticle. Increasing the concentration of beta-alanine to 2.0 M increases puncture-resistance further. Injection of 1.0 M of the beta-alanine analogue beta-aminoisobutyric acid, does not induce tanning or puncture-resistance, nor does injection of 1.0 M dopamine. However, injection of 1.0 M beta-alanine and 1.0 M dopamine increases puncture-resistance more than an injection of 1.0 M beta-alanine, though not more than an injection of 2.0 M beta-alanine alone. Within 10 min after injection of [3-³H]beta-alanine into newly eclosed normal flies, ³H becomes 8.7 times more concentrated in the cuticle than in an equal area of underlying epidermis. ³H is excluded from the epidermis or cuticle of ebony strains. Ebony strains show a deficiency of cuticular electron-absorbing material, and the cuticular lamellae show a tendency to separate from each other. Compaction of the chitinous lamellae is induced in alkali-detanned pupal sheaths by exposure to nascent quinones of N-acetyldopamine or N-beta-alanyldopamine. Glucosamine, but not N-acetylglucosamine, reacts with such quinones in tanning reactions. Under an infrared beam, black cuticular pigmentation induces more rapid heating of haemocoel fluids than does tan pigmentation. A theory of pigmentation and sclerotization relative to environmental adaptation is presented.     

Digenetic trematodes: quinone tanning system in eggshells

Three species of digenetic trematodes with colorless and transparent eggshells, Philophthalmus megalurus, Gorgoderina attenuata, and Megalodiscus temperatus, were compared by histochemical tests with Haematoloechus medioplexus, which has an identifiable quinone tanning system. Precursors for tanning including basic and tyrosine-rich proteins were identified histochemically in all species. Autoradiograms indicated that tyrosine was taken up after in vitro exposure of adult worms by vitelline tissue of G. attenuata and M. temperatus, but at a slower rate in the latter species. Protein synthesis inhibitor studies utilizing cycloheximide showed a reduction of tyrosine incorporation in the vitelline tissue of G. attenuata suggesting that tyrosine is incorporated into protein. Inhibition in M. temperatus was negligible, but evidence from other studies indicated absorption of cycloheximide by the adult may be minimal. Phenolase could not be found in G. attenuata and M. temperatus but was identified in P. megalurus. However, the enzyme system in the latter species seemed to be a partial one capable of oxidizing dihydroxyphenols but not monohydroxyphenols. Histochemical and solubility tests for keratin-type protein in the eggshells were inconclusive. It is suggested that positive identification of keratin as a structural protein of trematode eggshells be withheld until more reliable techniques can be employed. Possible evolutionary implications of the tanned eggshell in the Digenea are discussed.     

Use of the Phenol-Degrading <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain 21SG for Industrial Wastewater Decontamination

The features of a phenol-degrading Pseudomonas aeruginosa strain 21SG isolated from the soil at the territory of Russia’s largest manufacturing facility for synthetic tanning agents (city of Ufa, Bashkortostan Republic) are reported. The strain identification was based on cultural, morphological, physiological, biochemical, and morphometric features and on the results of comparative analysis of 16S rRNA gene sequences. The growth of P. aeruginosa 21SG was assessed in batch culture, and a decrease of phenol content in the culture liquid by 84% of the control level after 4 days was observed. The possibility of using P. aeruginosa 21SG for the destruction of phenol present in wastewater from the petrochemical industry and tanning extract production facilities was detected.     

Quinone methide sclerotization: A revised mechanism for β-sclerotization of insect cuticle

Molecular mechanisms responsible for the stiffening and tanning of insect cuticle are reviewed. Two mechanisms, viz., quinone tanning and β-sclerotization, both involving catecholamine derivatives as sclerotizing precursors, are known to strengthen the cuticle. Quinone tanning mechanism invokes the generation and reactions of o-benzoquinones as the sclerotizing agents, whereas β-sclerotization dictates the activation of catecholamine side chains prior to their incorporation into cuticle. The reactive intermediate for the latter process was proposed by other workers to be 1,2-dehydro-N-acetyldopamine and its quinone. The role of these two compounds in β-sclerotization is critically evaluated. Based on our observation that incubation of cuticular enzyme from Sarcophaga bullata with 4-alkylcatechols results in the production of soluble side chain oxygenated compounds and the formation of catechol-cuticle adducts, an alternate mechanism for β-sclerotization is proposed. This mechanism calls for the generation of quinone methides, tautomers of 4-alkyl-quinones, as the initial products of oxidation of catecholamine derivatives in cuticle. Quinone methides formed spontaneously react with available nucleophiles in cuticle, resulting in the generation of catechol-cuticle adducts and side chain hydroxylated products. Further oxidation of adducts and coupling to other structural units ensure crosslinking of cuticular components. The proposed quinone methide sclerotization accounts for all the chemical observations made on the β-sclerotized cuticle.     

Bursicon-mediated control of tanning in melanizing and non-melanizing first instar larvae of Schistocerca gregaria

Both melanization and sclerotization in first instar Schistocerca larvae are shown to have a common hormonal control mechanism. This hormone is shown to be analogous to bursicon, the tanning hormone of other insects, by the standard blowfly bioassay. The hormone is identical in both melanizing and non-melanizing first instar larvae of Schistocerca.     

Bursicon in the mealworm, Tenebrio molitor L. and its role in the control of postecdysial tanning

Using the adult Calliphora bioassay, we found that the tanning hormone, bursicon, is present in the blood of pupal and adult Tenebrio only at the time of ecdysis, when it is released massively from the thoracic and abdominal central nervous system. The hormone's half life in the blood is short (about 1–2 h). Contrary to the findings of other workers, we could find no evidence for the presence of the hormone in the haemolymph during pharate adult development, before ecdysis begins. When newly ecdysed pupae were ligated about the neck, adult development of the thorax and abdomen proceeded normally, but postecdysial tanning of the adult cuticle was almost completely prevented. This failure to tan was not due to lack of bursicon as the hormone was released normally in the ligated animals at the time of ecdysis. This suggests that a pre-ecdysial signal may be required for the development of epidermal competence to respond to bursicon.     

Beta-alanine and adaptation in Drosophila

Populations of Drosophila melanogaster show melanistic polymorphism. Adult melanization is inversely related to the rate of incorporation of beta-alanine into tanning cuticles. Light tan pigmentation is directly related to this incorporation. Beta-alanine induced tanning serves to protect pupae from ultraviolet light damage. Flies which do not fail to incorporate injected beta-alanine into developing cuticles, but which exhibit inhibited beta-alanine synthesis (flies homozygous for the gene ‘b’) are not only protected, as pupae, from ultraviolet damage by beta-alanine injection but also show enhanced mating success when injected as newly emerged adults. The metabolic block in this mutant appears in the pathway from orotic acid through uracil to beta-alanine.     

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.