Summary
The physical properties of insect cuticles are to a large extent determined by the degree of sclerotization (stabilization). The process of sclerotization often takes place shortly after eclosion, but may also occur before ecdy-sis or in connection with puparium formation. During sclerotization the two dopamine derivatives, N-acetyldo-pamine (NADA) and N-P-alanyldopamine (NBAD), are incorporated into the cuticular matrix. Incorporation of NBAD involves oxidation to its o-quinone followed by isomerization to a _/>-quinone methide, and both quinone derivatives can react with nucleophilic amino acids in the cuticular proteins. Incorporation of NADA involves oxidation to its o-quinone, isomerization to a _/>-quinone methide followed by isomerization to a,P-dehydro-N-acetyldopamine (dehydro-NADA) and oxidation of the latter to the unsaturated quinones, dehydro-NADA o-quinone and dehydro-NADA _/>-quinone methide. The dehydro-NADA />-quinone methide reacts readily with various nucleophilic groups, resulting in formation of inter-protein cross-links and polymers.
The pronounced diversity of insect cuticles indicates that several variations of the above-mentioned scheme are used for stabilizing cuticles, and a comparison of the details of the sclerotization processes occurring in different types of cuticle will probably be rewarding.
Introduction
The cuticle covers the insect body as an effective barrier between the animal and its surroundings; it provides protection against desiccation, microorganisms, and predators, and as an exoskeleton it provides attachment sites for muscles. Cuticle can occur as relatively hard and stiff regions, the sclerites, separated by more flexible and pliable cuticular regions, the arthrodial membranes, which make the various forms of locomotion possible. Marked differences in mechanical properties can be present on the microscopic level; two neighboring epidermal cells can produce cuticle with contrasting properties, indicating that cuticular composition is precisely controlled on the cellular level. The mechanical properties of the various cuticular regions are presumably optimal with respect to the forces to which they are exposed during the normal life of the animal; proper flight can only be sustained when the various wing regions have a near-optimal balance between stiffness and flexibility. If the wing material is locally too soft or too stiff, the varying air pressure during the wing strokes will not cause the wings to bend to the shapes needed for generating optimal lift.
The mechanical properties of cuticle are determined by the interplay of many factors, such as cuticular thickness, relative amounts of chitin and proteins, chitin architecture, protein composition, water content, intracuticular pH, degree of sclerotization, and other secondary modifications. Sclerotization of insect cuticle has been reviewed several times (Sugumaran, 1988, 1998; Andersen, 1990, 2005, 2010; Hopkins and Kramer, 1992; Andersen et al., 1996), but many aspects of the process are still poorly understood. The present review will attempt to give an up-to-date presentation of the sclerotization problems, and to draw attention to some of the problems that need to be investigated in more detail.
Cuticular sclerotization is a chemical process by which certain regions of the insect cuticle are transformed irreversibly from a pliant material into a stiffer and harder structure, characterized by decreased deformability, decreased extractability of the matrix proteins, and increased resistance to enzymatic degradation. During sclerotization the color of the cuticle may change; some cuticles remain nearly colorless, and some become lighter or darker shades of brown or black. The term "tanning" is often used synonymously with sclerotization, but sometimes it is specifically used for the processes whereby brownish (tan) cuticles are formed. Sclerotization often takes place in connection with molting, starting just after the new, as yet unsclerotized, cuticle has been expanded to its final size and shape, but some specialized cuticular regions are sclerotized while the insect is still in its pharate state inside the old cuticle. Such pre-ecdysially sclerotized regions cannot be expanded post-ecdysially, but help the insect to escape from the exuvium. The dipteran puparium is an example of a soft larval cuticle which is sclerotized at the end of the last larval instar to form a hard protective case, inside which metamorphosis to pupa and adult can take place. Sclerotization of structural materials in insects is not restricted to cuticle; other materials, such as egg cases, chorions, and silks, may be stabilized by chemical processes closely related to cuticular sclerotization.
A Model for Cuticular Sclerotization
During the past 70 years several models have been proposed for the chemical reactions occurring in the insect cuticle during the sclerotization process, and although many details of the individual steps in the reactions still are controversial or unexplored, there is general agreement concerning the main features of the process. The currently accepted sclerotization model is shown in Figures 1 and 2, and the main features are as follows: the amino acid tyrosine (1) is hydroxylated to 3,4-dihydroxyphenylalanine (DOPA, 2), which by decarboxylation is transformed to dopamine (3), a compound of central importance for both sclerotization and melanin formation. Dopamine can be N-acylated to either N-acetyldopamine (NADA, 4) or N-P-alanyldopamine (NBAD, 5), and both can serve as precursors in the sclerotization process. They are enzymat-ically oxidized to the corresponding o-quinones (6), which can react with available nucleophilic groups, whereby the catecholic structure is regained and the nucleophile is linked to the aromatic ring (11). The o-quinones of NADA and NBAD may also be enzymatically isomer-ized to the corresponding />-quinone methides (7), after which the P-position of the side chain can react with nucleophiles (12). The />-quinone methide of NADA can also be enzymatically isomerized to a side-chain-unsatu-rated catechol derivative, a,P-dehydro-N-acetyldopamine (dehydro-NADA, 8), but it is doubtful whether the />-quinone methide ofNBAD is isomerized to a,P-dehydro-N-P-alanyldopamine (dehydro-NBAD) to any significant extent.
Figure 1 Formation of sclerotization precursors NADA (4) and NBAD (5) from tyrosine (1).
Figure 2 Suggested scheme for formation of acyldopamine derivatives and concomitant formation of adducts during cuticular sclerotization. (6): o-quinone; (7): p-quinone methide; (8): dehydro-NADA; (9): dehydro-NADA o-quinone; (10): dehydro-NADA p-quinone methide; (11): C-6 substituted acyldopamine adduct; (12): p-substituted acyldopamine adduct. (13): dihydroxyphenyl-dihydrobenzodioxine derivative.
Dehydro-NADA can be oxidized to the corresponding o-quinone (9) and />-quinone methide (10). The latter can readily react with available catechols to give dihydroxyphenyl-dihydrobenzodioxine derivatives (13), and it will probably also react with nucleophilic amino acids to give various substitution products.
The cuticular nucleophilic groups which can react with the quinones formed from NADA and NBAD include the histidine imidazole group and free amino groups, such as terminal amino groups in proteins, e-amino groups in lysine residues, and the amino group in P-alanine. The phenolic group of tyrosine may furthermore react with the _/>-quinone methide of dehydro-NADA. The quinones may react with water and possibly also with hydroxyl groups and free amino groups in chitin, although little evidence exists for reactions with chitin. In vitro incubations have shown that the quinones may also react with available catechols. The various reactions between quinones and nucleophilic residues in the cuticular proteins result in the proteins being more or less covered by aromatic residues depending upon the degree of sclero-tization; some of the quinones may be involved in cross-linking the cuticular proteins and perhaps also in forming links between proteins and chitin, and some will only be linked to a single protein molecule, thereby increasing its hydrophobicity without being part of a covalent cross-link. During sclerotization, most of the water-filled spaces between the matrix proteins in the presclerotized cuticle will be filled with polymerized catecholic material. As a result of these processes the interactions between the cuticular components become stronger, the peptide chains become more difficult to deform, and the proteins can no longer move relative to each other or to the chitin system. Together, all these changes contribute to making the material stiffer and more resistant to degradation.
The various reactions involved in this model will be discussed in more detail in the following sections, with the main emphasis on aspects where the evidence is insufficient or missing, or where some observations disagree with the scheme, to indicate the areas where more research is needed. The appearance and properties of cuticle from different body regions of the same animal vary widely, and a considerable part of this variation, such as differences in coloration and mechanical properties, is probably due to quantitative and qualitative differences in the sclerotiza-tion process. There is no compelling reason to expect that exactly the same detailed process is used for stabilization in all types of solid cuticle, and generalizations based upon results obtained with a single or a few insect species can easily be misleading. Many cuticular types will have to be analyzed to help us understand how the individual steps involved in sclerotization can be modified to give the local variations between cuticular regions in a given insect, and between cuticles from different insect species. It will also be important to study how the reactions are controlled to give the optimal degree of sclerotization. Most of the results and ideas presented in this topic have been obtained by studies involving material from relatively few insect species, such as cuticle from blowfly larvae and puparia, cuticle from pupae of the moth Manduca sexta, and locust femur cuticle, and detailed studies of cuticle from other species will probably result in a much more varied and fascinating picture of cuticular sclerotization than is presented here.
Sclerotization (Tanning) Precursors
The terms "sclerotization agents" and "tanning agents" were originally used for the compounds which are secreted from the epidermal cells into the cuticle, where they are enzymatically oxidized to products sufficiently reactive to form covalent links to proteins and chitin. There is now a tendency to restrict the term "sclerotization agents" to the reactive species directly involved in forming links to the cuticular components. The compounds secreted from the epidermis to be activated in the cuticle shall accordingly be called "sclerotization precursors" (Sugumaran, 1998).
N-Acetyldopamine and N-p-Alanyldopamine
The first discovered and most common precursor for cutic-ular sclerotization is NADA (4), which is synthesized by N-acetylation of dopamine. The central role of NADA in sclerotization was demonstrated by Karlson’s research group (for review, see Karlson and Sekeris, 1976). They showed that NADA is incorporated in the puparial cuticle of the blowfly Calliphora vicina during its sclerotization, and that radioactively labeled tyrosine is metabolized to NADA when injected into last-instar larvae shortly before pupar-ium formation, and degraded when injected into younger larvae. The rate-limiting step was shown to be the enzy-matically catalyzed decarboxylation of DOPA controlled by the steroid hormone ecdysone (Karlson and Sekeris, 1962; Fragoulis and Sekeris, 1975). NADA was shown to be involved in cuticular sclerotization in several other insect species, such as the desert locust, Schistocerca gregaria (Karlson and Schlossberger-Raecke, 1962; Schlossberger-Raecke and Karlson, 1964). Incorporation of NADA into cuticle can be a very efficient process; after injection of radioactive NADA into young adult locusts, about 80% of the total radioactivity could later be recovered from the sclerotized cuticle (Andersen, 1971). NADA appears to be involved in cuticular sclerotization in all insect species investigated.
The amino acid P-alanine was reported as a constituent of several types of sclerotized cuticle (Karlson et al., 1969; Bodnaryk, 1971; Hackman and Goldberg, 1971; Srivastava, 1971), and it was suspected to participate in the sclerotization process (Andersen, 1979a). Hopkins et al. (1982) showed that the P-alanyl derivative of dopa-mine, NBAD (5), is a sclerotizing precursor in the cuticle of M. sexta pupae, thus accounting for the presence of P-alanine in hydrolysates of the fully sclerotized cuticle. NBAD is also a sclerotization precursor in the other cuticles from which P-alanine can be released by acid hydrolysis, such as the cuticle of the red flour beetle, Tribolium castaneum (Kramer et al., 1984). The synthesis and utilization of NBAD during pupation of M. sexta have been reported (Krueger et al., 1989).
The first step in the synthesis of NADA and NBAD is hydroxylation of tyrosine to DOPA (2); this process can be catalyzed both by the o-diphenoloxidase, tyrosi-nase, which can also catalyze the oxidation of DOPA and other o-diphenols to o-quinones, and by the enzyme tyrosine hydroxylase. Tyrosinases play an important role in the defense systems of insects, and tyrosine hydroxy-lase appears to be the enzyme responsible for synthesis of the sclerotization precursors. The enzyme is present in epidermal cells, and both hardness and coloration of T. castaneum adult cuticle are significantly diminished when the activity of the enzyme is reduced by RNA interference (Gorman and Arakane, 2010).
Tyrosine hydroxylase is also important for sclerotiza-tion and darkening of the adult cuticle of D. melanogas-ter; it has been demonstrated that production of tyrosine hydroxylase in the epidermal cells in the pharate adult fruit fly is induced by the neurohormone CCAP (crustacean cardioactive peptide), and soon after ecdysis the sclerotization process is initiated by the neurohormone bursicon, which induces activation of the tyrosine hydroxyl-ase by phosphorylation of a serine residue (Davis et al., 2007). It is interesting that no such phosphorylation of tyrosine hydroxylase is needed for initiation of sclerotization of the puparium of D. melanogaster, a process that appears to be controlled by release of tyrosine from O-phoshotyrosine (Davis et al., 2007). The hormone bursi-con has functions other than induction of sclerotization, such as plasticization and stretching of wings after ecdy-sis, formation of melanin, and deposition of endocuticle (Dai et al., 2008; Honegger et al, 2008).
The DOPA residues formed in the epidermal cells are not directly involved in sclerotization, but are transformed to dopamine (3) by a DOPA-decarboxylase, and at least in T. castaneum this decarboxylase is essential for production of both NADA and NBAD (Arakane et al, 2009). Another important decarboxylase in the epidermal cells is an aspartate-1-decarboxylase, which transforms aspartic acid to P-alanine. Reducing its activity by means of RNA interference resulted in adult beetles with a completely black cuticle instead of the rust-red cuticle of control animals. The black beetles contained less NBAD and more dopamine than untreated animals, and formation of the black color (melanins) could be prevented by injection of P-alanine. Mechanical tests of the elytra of black animals indicated that the cuticle was less cross-linked than in the controls (Arakane et al., 2009).
The N-acetyldopamine synthetase, which acetylates dopa-mine to NADA, and the N-P-alanyldopamine synthetase, which P-alanylates dopamine to NBAD, are present in epidermal cells beneath sclerotizing cuticle (Krueger et al., 1989; Wappner et al., 1996; Perez et al., 2002), and it appears that insect epidermis is equipped with all the enzymes necessary for producing the sclerotization precursors. The acylation reactions can also occur in tissues other than epidermis; for instance, both NADA and NBAD can be produced within the nervous system (Krueger et al., 1990).
The two sclerotizing compounds, NADA and NBAD, are used together in many cuticular types, but the cuticle of some insects, such as the locusts Schistocerca gregaria and Locusta migratoria, appears to be exclusively sclerotized by NADA, and no P-alanine has been obtained from their acid hydrolysates. No cuticle has yet been reported to be sclerotized exclusively by NBAD. A correlation has been reported between the intensity of brown color of the fully sclerotized cuticle and the amounts of NBAD taking part in the sclerotization process: cuticles that are sclerotized exclusively by NADA are often colorless or very lightly straw-colored, and when NBAD dominates in the process dark brown cuticles are formed (Brunet, 1980; Hopkins et al., 1984). Czapla et al. (1990) reported that cuticular strength in five differently colored strains of the cockroach Bla-tella germanica correlated well with the concentrations of P-alanine and NBANE, and melanization correlated with dopamine concentration. During sclerotization, cuticular strength, as well as cuticular concentrations of P-alanine and NBAD, increased more rapidly in the rust-red wild type of T. castaneum than in the black mutant strain, whereas cuticular dopamine increased more rapidly in the black mutant than in the wild type (Roseland et al., 1987).
Significant amounts of sclerotization precursors are often present as conjugates before the onset of sclerotization. The conjugates can be glucosides, phosphates, or sulfates (Brunet, 1980; Kramer and Hopkins, 1987), they are not easily oxidized and have to be hydrolyzed to free catechols before they can take part in sclerotization. It is assumed that the catechol conjugates serve as a storage reservoir of catecholamines ready to be used when the need for sclerotization arises (Brunet, 1980). A dopamine conjugate, identified as the 3-O-sulfate ester, is present in the hemolymph of newly ecdysed cockroaches, and its concentration decreases rapidly during sclerotization of the cockroach cuticle. The sulfate moiety is not transferred into the cuticle; removal of sulfate and acylation of the liberated dopamine to NADA and/or NBAD will most likely take place in the epidermal cells (Bodnaryk and Brunet, 1974; Czapla et al, 1988, 1989).
Hopkins et al. (1984) reported that a large fraction of the various catecholamines in M. sexta hemo-lymph and cuticle is present as acid labile conjugates. In larval and pupal hemolymph these conjugates are mainly 3-O-glucosides together with small amounts of 4-O-glucosides, whereas adult hemolymph contains more of the 4-O-glucoside than of the 3-O-glucoside (Hopkins et al., 1995). Both conjugated and unconjugated forms of NADA and NBAD are present in M. sexta hemo-lymph, but are only present in low amounts in the cuticle (Hopkins et al., 1984), indicating that the epidermal cells contain a P-glucosidase able to hydrolyze the conjugates to unconjugated catecholamines and glucose.
Putative Sclerotization Precursors
So far, convincing evidence that they function as cuticu-lar sclerotization precursors has only been obtained for NADA and NBAD, but other compounds have been described as likely sclerotization precursor candidates, such as N-acetyl-norepinephrine (NANE) (14), N-P-alanyl-norepinephrine (NBANE) (15), and 3,4-dihydroxyphenylethanol (DOPET) (16) (Figure 3). Probably, they all have some role in sclerotization, but their formation and metabolism needs to be studied in more detail.
N-acetylnorepinephrine (NANE) and N-fi-alanylnorepinephrine (NBANE) NANE and NBANE are special cases among cuticular catechols, because they can be considered as both by-products of the sclerotization process and precursors for sclerotization. They have been reported to occur both free and as O-glucosides in hemolymph and integument in several insects (Hopkins et al., 1984, 1995; Morgan et al., 1987; Czapla et al, 1989). NANE and NBANE can be generated within the cuticle, when the enzymatically produced />-quinone methides of NADA and NBAD react with water instead of either reacting with cuticular proteins or isomerising to dehydro-derivatives, and they can also be produced by hydrolysis of unidentified labile products of the sclerotization process. Mild acid treatment of sclerotized cuticle can release NANE and NBANE from the cuticular structure, probably due to hydrolysis of a bond between the P-position of the catechols and some cuticular constituent. The nature of the bond is uncertain, but it could be an ether linkage connecting the acyldopamine side chain to chitin. Formation of an ether, P-methoxy-NADA, occurs when isolated pieces of cuticle or extracted cuticular enzymes act upon NADA in the presence of methanol; the compound is acid labile, and is readily hydrolyzed to free NANE (Andersen, 1989a; Sugumaran et al., 1989a).
Figure 3 Hypothetical sclerotization precursors. (14): W-acetyl-norepinephrine (NANE); (15): W-P-alanyl-norepinephrine (NBANE); (16): 3,4-dihydroxyphenylethanol (DOPET); (17): gallic acid.
NANE can be covalently incorporated into the cuticu-lar matrix during sclerotization, indicating that the compound can serve as a sclerotization precursor (Andersen, 1971), and this is probably also the case for NBANE. When radioactively labeled NANE was injected into newly ecdysed locusts a significant fraction of the radioactivity (about 15%) was incorporated into the cuticle, and hydrolysis was needed to release the activity. Acid hydrolysis of cuticle from locusts injected with labeled norepinephrine resulted in the release of both labeled nor-epinephrine and arterenone, whereas little radioactivity was present in the neutral ketocatechol fraction. This is in contrast to parallel experiments where labeled dopamine was injected into locusts and nearly all the radioactivity was recovered as neutral ketocatechols, indicating that the cuticular enzymes can catalyze the incorporation of nor-epinephrine and NANE into the cuticular matrix, but not as efficiently and not by the same route as incorporation of dopamine and NADA.
Dihydroxyphenylethanol (DOPET) The third putative sclerotization precursor, 3,4-dihydroxy-phenylethanol (DOPET, 16), is present in the hemolymph and integument of some insects; it has been obtained from cuticle of the cockroach Periplaneta americana (Atkinson et al., 1973a; Czapla et al., 1988) and the beetle Pachynoda sinuata (Andersen and Roepstorff, 1978), and can function as substrate for the cuticular phenoloxidases. Extraction and acid hydrolysis of sclerotized cuticles have yielded various DOPET derivatives, suggesting that DOPET can be transported into the cuticle and incorporated into the cuticular matrix during sclerotization. Adducts of DOPET and histidine have been obtained by acid hydrolysis of sclerotized Manduca pupal cuticle and identified by means of mass spectrometry (Kerwin et al., 1999), and a dihydroxyphenyl-dihydrobenzodioxine-type adduct of DOPET and NADA was extracted from sclerotized beetle (P. sinuata) cuticle (Andersen and Roepstorff, 1981), but the metabolism of DOPET in insects needs to be studied in much more detail.
The relative roles of dopamine, NANE, NBANE, and DOPET compared to the two major sclerotization precursors, NADA and NBAD, have never been properly established. The compounds are probably only of minor importance for the mechanical properties of sclerotized cuticles, but their involvement in the sclerotization process could play a role in fine-tuning of the cuticular properties.
Other sclerotization precursors It has been reported that improved growth of the tree locust Anacridium melanorhodon can be obtained by addition of gallic acid (17) (Figure 3) and other plant phenols to its food, and that the ingested plant phenols are incorporated into the cuticle and may contribute to its stabilization (Bernays et al., 1980; Bernays and Woodhead, 1982).
Transport of Sclerotization Precursors to the Cuticle
The cuticle is constantly exposed to external forces varying in intensity and direction during insect movements; the mechanical properties of the cuticular regions must correspond to the forces they are exposed to, and to guarantee this a local regulation of the degree of sclerotization must be present. The sclerotization can be regulated via availability of sclerotization precursors, for instance by control of local synthesis of precursors and their uptake from hemolymph into epidermis, and by control of the transport of precursors from epidermal cells to cuticular matrix, where they will be exposed to the enzymes converting them to sclerotization agents. Precursors for scler-otization can be synthesized by the epidermal cells, but they may also be produced by other cell types, such as hemocytes and/or fat body cells.
The white pupa mutant of the Mediterranean fruit fly, Ceratitis capitata, does not sclerotize its puparium, but normal larval and adult cuticles are produced. The concentrations of the various catecholamines are very low in the mutant puparial cuticle compared to the wild type strain, whereas the concentrations in the hemolymph of NADA, NBAD, and dopamine are about 10 times higher in the mutant than in the wild type, indicating that the mutant is defective in a system transporting catechol-amines from hemolymph to puparial cuticle (Wappner et al., 1995). Precursors, such as dopamine, NADA, and NBAD, injected into the hemocoel of locusts shortly before or during cuticular sclerotization, are rapidly taken up by the epidermal cells and transported into the cuticle, whereas precursors injected several days before the start of sclerotization are mainly degraded or modified by gly-cosylation or phosphorylation. Such precursor conjugates may either remain in the animal to serve as a reserve pool of sclerotizing material, or be excreted via the Malphigian tubules by the standard detoxification mechanisms. The different fates of NADA injected before or during ecdy-sis could be explained by different activities of transport systems located in the apical or basolateral membranes of the epidermal cells.
It is thus likely that the epidermal cells possess specific transport systems controlling transfer of the right amounts of sclerotizing precursors into the cuticle at the right time, and presumably such transport systems are present in the basolateral cell membrane to facilitate uptake from the hemolymph and in the apical cell membrane to control the transport of sclerotizing precursors from the cells into the cuticle. Active diphenoloxidases (laccases) are, in some insects, present in the unsclerotized pharate cuticle (Andersen, 1972a, 1979b, and unpublished data), and presumably the precursors are only transported into these cuticles when sclerotization is initiated following ecdysis. Is activation of post-ecdysial transport of sclerotization precursors into the cuticle governed by the presence of the hormone bursicon?
In cuticles sclerotized before ecdysis, the sclerotization precursors might be transported without delay from the epidermal cells into the pharate cuticle to be oxidized and incorporated. It is not known whether the pre-ecdysial transport occurs via specific transporters in the apical membrane or whether it happens by passive diffusion.
It has been reported that catechol derivatives attached to proteins can be transported intact from hemolymph to the cuticular structure to serve as combined matrix components and sclerotization precursors.Such transport indicates that receptors able to recognize the catechol—protein complexes are present in the membranes of the epidermal cells.
