Cuticular Tanning Research Articles

Alternative splicing of POUM2 regulates embryonic cuticular formation and tanning in Bombyx mori.

Insect cuticle is an apical extracellular matrix produced by the epidermis, tracheal, hind- and foregut epithelia during embryogenesis and renewed during molting and metamorphosis. However, the underlying regulatory mechanism for embryonic cuticle formation remains largely unclear. Here, we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori, a model lepidopteran insect. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein-9-mediated knockout of POUM2 resulted in the defect of cuticular deposition, pigmentation, and sclerotization in the embryos. Differentially expressed transcripts analysis of 7-d-old embryos identified 174 up- or downregulated cuticular protein transcripts, 8 upregulated chitin degradation transcripts, 2 downregulated chitin synthesis transcripts and 48 up- or downregulated transcription factor transcripts in the POUM2-/- embryos. The expression levels of the key factors of the tyrosine metabolic pathway, such as tyrosine hydroxylase (Th), Dopa decarboxylase (DDC), and arylalkylamine N-acetyltransferase (aaNAT), were significantly decreased in the POUM2-/- embryos. POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element (CRE) in the Th promoter and increased the transcription of Th, whereas POUM2-S could not bind the POU CRE, although it also increased the transcription of Th. Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA (messenger RNA) and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA. These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism, and the cuticular pigmentation and sclerotization by regulating tyrosine metabolism during embryogenesis. This study provides new insights into novel function of POUM2 in embryogenesis.

Glycosyltransferases: the multifaceted enzymatic regulator in insects.

Glycosyltransferases (GTs) catalyse the reaction of glyco-conjugation of various biomolecules by transferring the saccharide moieties from an activated nucleotide sugar to nucleophilic glycosyl acceptor. In insects, GTs show diverse temporal and site-specific expression patterns and thus play significant roles in forming the complex biomolecular structures that are necessary for insect survival, growth and development. Several insects exhibit GT-mediated detoxification as a key defence strategy against plant allelochemicals and xenobiotic compounds, as well as a mechanism for pesticide cross-resistance. Also, these enzymes act as crucial effectors and modulators in various developmental processes of insects such as eye development, UV shielding, cuticle formation, epithelial development and other specialized functions. Furthermore, many of the known insect GTs have been shown to play a fundamental role in other physiological processes like body pigmentation, cuticular tanning, chemosensation and stress response. This review provides a detailed overview of the multifaceted functionality of insect GTs and summarizes numerous case studies associated with it.

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.

Synthesis and Antityrosinase Activities of Alkyl 3,4-Dihydroxybenzoates

In insects, tyrosinase plays important roles in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. Thus, tyrosinase may be regarded as a potential candidate for novel bioinsecticide development. A family of alkyl 3,4-dihydroxybenzoates (C₆-C₉), new tyrosinsase inhibitors, were synthesized. Their inhibitory effects on the activity of tyrosinase have been investigated. The results showed all of them could inhibit the activity of tyrosianse effectively. The order of potency was nonyl 3,4-dihydroxybenzoate (C₉DB) > octyl 3,4-dihydroxybenzoate(C₈DB) > heptyl 3,4-dihydroxybenzoate(C₇DB) > hexyl 3,4-dihydroxybenzoate (C₆DB). The kinetic analysis of these four compounds on tyrosinase was taken to expound their inhibitory mechanism. The research of the control of insects in agriculture was taken as C₆DB for example. C₆DB could inhibit the development and molting of Plutella xylostella effectively. To clarify its insecticidal mechanism, we researched the expression of tyrosinase in the P. xylostella treated with C₆DB by real-time quantitative PCR. The results showed C₆DB could inhibit the expression of tyrosinase in the P. xylostella as expected.

RNA Interference as a Method for Target-Site Screening in the Western Corn Rootworm,Diabrotica Virgifera Virgifera

To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets.

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene ( CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.

A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning inDrosophila melanogaster

A neuropeptide hormone-signalling pathway controls events surrounding eclosion in Drosophila melanogaster. Ecdysis-triggering hormone, eclosion hormone and crustacean cardioactive peptide (CCAP) together control pre-eclosion and eclosion events, whereas bursicon, through its receptor rickets (RK), controls post-eclosion development. Cuticular tanning is a convenient visible marker of the temporally precise post-eclosion developmental progression, and we investigated how it is controlled by the ecdysis neuropeptide cascade. Together, two enzymes, tyrosine hydroxylase (TH, encoded by ple) and dopa decarboxylase (DDC, encoded by Ddc), produce the dopamine that is required for tanning. Levels of both the ple and Ddc transcripts begin to accumulate before eclosion, coincident with the onset of pigmentation of the pharate adult bristles and epidermis. Since DDC activity is high before the post-eclosion onset of tanning, a different factor must be regulated to switch on tanning. Transcriptional control of ple does not regulate the onset of tanning because ple transcript levels remain unchanged from 24 hours before to 12 hours after eclosion. TH protein present before eclosion is degraded, and no TH activity can be detected at eclosion. However, TH protein rapidly accumulates within an hour of eclosion and we provide evidence that CCAP controls this process. Furthermore, we show that TH is transiently activated during tanning by phosphorylation at Ser32, as a result of bursicon signalling. We conclude that the ecdysis hormone cascade acts as a regulatory switch to control the precise onset of tanning by both translational and activational control of TH.

Anatomy and functions of brain neurosecretory cells in diptera.

In the larval brain of dipteran insects, there are two medial and three lateral groups of neurons innervating the ring gland. One lateral group extends fibers to the corpus allatum. After metamorphosis, a large cluster of the medial group in the pars intercerebralis and two lateral groups in the pars lateralis innervate the retrocerebral complex and some neurons from the lateral group and a few from the medial group extend fibers to the corpus allatum in the adults. Neuropeptides such as insulin-like peptides, FMRFamide related peptides, Locusta-diuretic hormone, beta-pigment dispersing hormone, Manduca sexta-allatostatin, ovary ecdysteroidogenic hormone, and proctolin have been immunocytochemically revealed in medial groups in the pars intercerebralis, and FMRFamide related peptides, beta-pigment dispersing hormone, corazonin, and M. sexta-allatostatin in lateral groups in the pars lateralis of dipteran brains. In mosquitoes after the blood meal, ovary ecdysteroidogenic hormone from 2-3 pairs of medial neurosecretory cells is released at the corpus cardiacum to stimulate the ovaries to secrete ecdysteroid to cause ovarian development. In addition to ovarian development, removal and implantation experiments have shown that neurosecretory cells in the pars intercerebralis and pars lateralis are involved in control of reproductive diapause, cuticular tanning, sugar metabolism, and diures.

Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedesaegypti

In this study we describe the purification and molecular cloning of a dopachrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti. DCE catalyzes the conversion of l-dopachrome to 5,6-dihydroxyindole in the melanization pathway. Melanin biosynthesis is involved with crucial protective phenomena in mosquitoes, including egg chorion and cuticular tanning, wound healing, and the melanotic encapsulation immune response. The enzyme was purified to homogeneity by various chromatographic techniques from A. aegypti larvae and has a relative molecular mass of 51 kDa as-revealed by SDS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7.5–8.0 and substrate activity for l-dopachrome and aminochromes generated from dopa methyl ester, α-methyl dopa and dopamine. Trypsin digestion of the isolated DCE and subsequent reverse-phase separation resulted in the isolation of several polypeptide fragments, from which two partial internal amino acid sequences were obtained by Edman degradation. PCR amplification, using a degenerate primer based on one internal amino acid sequence and an oligo-dT primer, produced a 650 bp DNA fragment. Subsequent screening of an A. aegypti pupal cDNA library resulted in the isolation of a 1.6 kb clone containing coding sequence for both internal DCE amino acid sequences, thereby confirming the identity of the isolated gene product (pAaDce1) as DCE. Northern analysis revealed the constitutive expression of DCE message in developmental stages and adults, with the majority of transcript localized in the fat body and ovaries of adult females. AaDce1 mRNA increased in abundance above constitutive levels in adult females when a melanotic encapsulation immune response was initiated by the intrathoracic inoculation of Dirofilaria immitis microfilariae.

Changes in catecholamine levels in the gut and frass of the corn earworm, Helicoverpa zea induced by dietary l-azetidine-2-carboxylic acid

Several studies have shown that a plant-derived lower homologue of proline, l-azetidine-2-carboxylic acid (AZC), fed to larvae of the corn earworm, Helicoverpa zea, induces toxicity leading to an array of developmental defects including incomplete pupal cuticular sclerotization or tanning. Destabilized cuticular tanning has recently been shown to be correlated with a deficiency in a hemolymph catecholamine, N-β-alanyldopamine (NBAD) (Okot-Kotber and Adeyeye, 1995). Analysis of gut and frass catecholamine contents has revealed that an AZC-supplemented diet induces a two-fold increase in the excretion of dopamine (DA) and β-alanine. Also, more 3,4-dihydroxyphenylalanine (DOPA) was excreted in this group than in the controls, although overall, less DOPA was detected than DA in both cases. Four catechols of unknown structure, designated FC1, FC2, FC3 and FC4 according to their relative chromatographic retention times, were found in frass extracts with relatively higher levels in the AZC-treated larvae than in the controls. Three of these, FC1, FC2 and FC3 were also found in the diet, but FC4 was detected only in frass. Gut tissue catecholamines were much higher (by approximately 100-fold) than found in the frass. Approximately two-fold higher DOPA and seven-fold higher DA concentrations were detected in the guts of pupae with incomplete tanning, that had developed from AZC-treated larvae, compared to the controls. It appears, therefore, that the deficiency in NBAD observed in the hemolymph of AZC-fed larvae is largely caused by a slower rate of acylation of DA into NBAD, which results in the accumulation of DA in the gut. The data suggests that there is a defective β-alanyldopamine synthetase in the AZC-treated insects rather than an increased catabolism of NBAD.

Dopachrome conversion activity in Aedes aegypti: Significance during melanotic encapsulation of parasites and cuticular tanning

Phenol oxidase (PO) and dopachrome conversion enzyme (DCE) were partially purified from Aedes aegypti larvae by ammonium sulfate fractionation. PO from A. aegypti functions in the hydroxylation of monophenols (e.g., tyrosine and tyramine) to their related o-diphenols, and the oxidation of o-diphenols (e.g., l-dopa, dopamine, N-acetyldopamine) to their respective o-quinones. Partially purified DCE showed high specificity toward dopachrome generated from dopa with the l-configuration. The combined effects of PO and DCE significantly accelerated melanization pathways when l-dopa was used as substrate. Significant DCE activity also was detected in hemolymph samples from adult, female A. aegypti, and undoubtedly plays a role in melanotic encapsulation reactions.

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.

Hormonal Control of Transmitter Plasticity in Insect Peptidergic Neurons: I. Steroid Regulation of the Decline in Cardioacceleratory Peptide 2 (Cap2) Expression

Transmitter plasticity, the ability to alter transmitter expression, has been documented in several different preparations both in vivo and in vitro. One of these is the tobacco hawkmoth, Manduca sexta, whose central nervous system contains four individually identified lateral neurosecretory cells (LNCs) that undergo a postembryonic transmitter switch in vivo. In larvae, the LNCs express high levels of a myoregulatory peptide, cardioacceleratory peptide 2 (CAP2). In contrast, the predominant LNC transmitter in adult moths in bursicon, a classic insect peptide hormone responsible for cuticular tanning. Here we show that the CAP2-to-bursicon conversion by the LNCs is a multi-step process beginning with a decline in CAP2 levels midway through the final larval stage. We provide several lines of evidence that this CAP2 drop is regulated by the insect steroid hormone 20-hydroxyecdysone (20-HE). The LNCs exhibit a fall in CAP2 levels at the beginning of metamorphosis, immediately after the commitment pulse of 20-HE when steroid levels are elevated. LNCs not exposed to this 20-HE rise do not exhibit a decline in CAP2 level. The transmitter switch can also be prevented by using an analog of juvenile hormone to create a larval hormonal environment during the commitment pulse of 20-HE. The CAP2 decline in the LNCs could be directly induced by exogenous steroid application, but only under conditions where the LNCs remained connected to the brain. Thus, the first step in the transmitter switch by the LNCs, the decline in CAP2 levels, is triggered by the commitment pulse of 20-HE, which may act indirectly through a set of steroid-sensitive cells in the brain.

Hormonal control of transmitter plasticity in insect peptidergic neurons. II. Steroid control of the up-regulation of bursicon expression.

Each abdominal ganglion of the central nervous system of the tobacco hawkmoth, Manduca sexta contains four individually identified lateral neurosecretory cells (LNCs) that undergo a postembryonic transmitter switch in vivo. In the embryonic and caterpillar stages, the primary LNC transmitter is cardioacceleratory peptide 2 (CAP2), a myoregulatory peptide. During metamorphosis, these cells stop expressing CAP2 and instead produce bursicon, a classic insect peptide hormone responsible for cuticular tanning. We have previously reported that this transmitter plasticity is under the control of the insect steroid hormone 20-hydroxyecdysone (20-HE), which surges twice during the last larval instar. In that report we showed that the CAP2 decline is indirectly regulated by the first 20-HE rise, the commitment pulse (CP). Here we provide evidence that the rise in bursicon levels in the LNCs is directly triggered by the second 20-HE surge, the prepupal peak (PP). We performed several experimental manipulations that exposed LNCs to the PP without the CP; cells treated in this manner exhibited a significant rise in bursicon content. In contrast, bursicon levels remained unchanged in those LNCs exposed only to the CP. Exposure to the PP triggered a precocious increase in bursicon expression in LNCs from the penultimate larval stage. Increased bursicon levels in the LNCs were also induced by direct infusion of 20-HE. Taken together, the results of these experiments suggest that the rise in bursicon in the LNCs during metamorphosis is due to the direct action of the PP on the LNCs. Thus, the two 20-HE surges combine to regulate the CAP2-to-bursicon switch in the LNCs, the first acting indirectly to cause a decline in CAP2 levels and the second triggering a rise in bursicon expression, possibly by a direct action on the LNCs.

The Drosophila melanogaster stranded at second (sas) gene encodes a putative epidermal cell surface receptor required for larval development

Several lethal mutations were identified previously in the 84BD interval of the Drosophila melanogaster third chromosome (Lewis et al., 1980; Cavener et al., 1986b). We have examined the l(3)84Cd complementation group and found that mutants exhibit novel cuticular defects and die during larval development. The lethal phase occurs during the first larval molt or subsequently during the second instar larval stage; hence, we have named the gene stranded at second (sas). There are no apparent effects on the rate of development of embryos or first instar larvae. Second instar larvae which survive the molt exhibit a marked reduction in growth and eventually die as small second instar larvae. Incomplete penetrance in some weak sas alleles can yield fertile adults. In addition to the lethal phenotype, a segmentally repeated pattern of tanned spots is found within the ventral setal belts of mutant larvae. The position of the spots is always either between the fourth and fifth row of setae (cuticular projections) or between the first and second row of setae. The spots are adjacent to the muscle attachment sites in the setal belt region. Another common larval phenotype is the abnormal tanning of the ventral surface of the pharynx. The sas gene was cloned, and both the cuticular tanning and the larval lethal phenotypes were complemented by P-element-mediated transformation with a genomic DNA-cDNA construct. Three major sas transcripts are expressed throughout development in cuticle secreting epidermal tissues. The sas transcripts show stage- and tissue-specific patterns of expression with switches in transcript patterns occurring at the molts. The inferred 1348-amino-acid sequence suggests that sas encodes a cell surface protein which functions as a receptor. The putative extracellular region contains four tandem repeats of a cysteine-rich motif which is similar to a cysteine pattern present in procollagen and in thrombospondin. Following this region are at least three copies of a fibronectin type III class repeat. The short (35 amino acids) intracellular domain contains a sequence (NPXY) that has been implicated in endocytosis via coated pits.

Evolution of the cuticular pigmentation as a system for developmental timing for Panstrongylus megistus and Rhodnius prolixus adults

A system for developmental timing for Panstrongylus megistus and Rhodnius prolixus (Hemiptera: Reduviidae) adults is presented, using as a marker, the evolution of the cuticular tanning after ecdysis. In both species the setting of the cuticular pigmentation pattern after imaginai moult is a homogeneous process in population, i.e. no individual variations were detected. The system allows one to estimate the exuvial time up to 5 or 7 hr after occurrence. A preferential exuvial period (PEP) was determined in the population under usual rearing conditions by using this system. The PEP runs from 2100 to 0500 hr for P. megistus and from 0100 to 0800 hr for R. prolixus. These data are meaningful for endocrine studies on those species. Studies on PEP under natural environmental conditions would be useful for the control of Chagas’ disease vectors.

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.

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.

β-alanine tanning of Drosophila cuticles and chitin

The cuticular lamellae of the Drosophila melanogaster mutants, “ebony” (which fails to transport β-alanine into the cuticle during development) and “black” (which shows inhibited β-alanine synthesis) are wider and more diffuse than normal, and the endocuticular microfibrils lack distinctness. Injection of β-alanine into “black” induces normal compact lamellation and cuticular tanning. β-alanine readily forms tan colours when reacting with pure chitin or its saccharide monomer, N- acetyl-glucosamine (or glucose). Other dipolar omega amino acids do likewise. β-alanine is bound to pure chitin more rapidly than is l-alanine. Pre-treatment of chitin with β-alanine depresses subsequent binding of DOPA. Complexing of β-alanine with mucopolysaccharides or glycopolysaccharides may be responsible for inhibition of catabolism of glucose. Such inhibition is observed in Ehrlich ascites tumor cells in vitro.

Possible involvement of cockroach haemocytes in the storage and synthesis of cuticle proteins

The injection of UL 14C-leucine into newly ecdysed immature cockroaches resulted in the labelling of both haemocyte and serum proteins. Serum proteins were purified by gel filtration, concentrated and reinjected into other freshly ecdysed animals. After incubations of one hour, radioactivity was detected in serum, haemocyte, and cuticle proteins. Similar experiments using labelled soluble blood cell proteins also produced radioactivity in the serum, cells and cuticle. The possible rôle of haemocytes in cuticle protein synthesis is denoted and its significance in regard to cuticular tanning is discussed.