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Cuticle formation and pigmentation in beetles
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ScienceDirect Cuticle formation and pigmentation in beetles Mi Young Noh1, Subbaratnam Muthukrishnan2, Karl J Kramer2 and Yasuyuki Arakane1 Adult beetles (Coleoptera) are covered primarily by a hard exoskeleton or cuticle. For example, the beetle elytron is a cuticle-rich highly modified forewing structure that shields the underlying hindwing and dorsal body surface from a variety of harmful environmental factors by acting as an armor plate. The elytron comes in a variety of colors and shapes depending on the coleopteran species. As in many other insect species, the cuticular tanning pathway begins with tyrosine and is responsible for production of a variety of melanin-like and other types of pigments. Tanning metabolism involves quinones and quinone methides, which also act as protein cross-linking agents for cuticle sclerotization. Electron microscopic analyses of rigid cuticles of the red flour beetle, Tribolium castaneum, have revealed not only numerous horizontal chitin-protein laminae but also vertically oriented columnar structures called pore canal fibers. This structural architecture together with tyrosine metabolism for cuticle tanning is likely to contribute to the rigidity and coloration of the beetle exoskeleton. Addresses 1 Department of Applied Biology, Chonnam National University, Gwangju 61186, Republic of Korea 2 Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, United States Corresponding author: Arakane, Yasuyuki ([email protected])
Current Opinion in Insect Science 2016, 17:1–9 This review comes from a themed issue on Molecular physiology Edited by Takema Fukatsu and Ryo Futahashi For a complete overview see the Issue and the Editorial Available online 12th May 2016 http://dx.doi.org/10.1016/j.cois.2016.05.004 2214-5745/# 2016 Elsevier Inc. All rights reserved.
and skeleton, protecting insects from environmental stresses and mechanical damage. The Coleoptera is the largest insect order, and its cuticle including that of the elytron (Box 1), which is a highly modified and tanned (sclerotized and pigmented) forewing, has been an attractive material for study not only because of its metallic and lustrous coloration but also due to its mechanical properties as a rigid and lightweight tissue. Here, we review the functional importance and the diversity of the genes involved in the tyrosine-derived cuticle tanning pathway, and the morphology and ultrastructure of rigid types of beetle cuticle. The functional importance, unique localization, and cross-linking of specific CPs in the formation of the exoskeleton are also discussed.
Beetle cuticle pigmentation Several kinds of chemical pigments such as melanins, pterins, ommochromes, antraquinones, aphins, tertapyrroles, carotenoids and flavonoids/anthocyanins contribute to a variety of insect colors [1]. However, there have been only a few studies focusing on the identification, biosynthesis, genetic regulation and function of these pigments in beetles. For example, carotenoid content is responsible for the variable orange-to-red coloration of elytra of the Asian ladybird beetle, Harmonia axyridis, and this red hue appears to be aposematically correlated with the content of their defensive alkaloid molecules [2]. Like other insect species, melanin-like and quinonoid pigments produced by tyrosine metabolism play a major role in the darkening of beetle cuticle [3,4,5,6]. Tyrosine metabolism is also critical for cuticle hardening (sclerotization). Cuticle tanning involving tyrosine metabolism has been a study of major emphasis in our laboratories.
Tyrosine-mediated cuticle pigmentation and sclerotization Introduction Insects, belonging to the most diverse and successful animal phylum Arthropoda, have developed and acquired superior adaptability to the natural environment, excellent communication systems, and optimized body designs and functions throughout their evolution. In addition to these features, the exoskeleton or cuticle gives the insects excellent capabilities that help to account for their evolutionary success. The insect cuticle is a remarkable biomaterial/biomass primarily formed from structural cuticle proteins (CPs) and the linear polysaccharide, chitin. This strong extracellular material serves both as a skin www.sciencedirect.com
Cuticle tanning (pigmentation and sclerotization) is a complex process, which includes hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and decarboxylation of DOPA to dopamine (Box 2). Additional steps for melanization include oxidation of DOPA and dopamine to dopa-quinone and dopamine-quinone, conversion of these quinones to dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), via intramolecular cyclization, oxidation of DHI and DHICA to DHI-chrome and DHICA-chrome (melanochromes) and then polymerization of the melanochromes to form melanin-like pigments. Pigmentation involving N-acylated quinones requires N-acetylation of dopamine to Current Opinion in Insect Science 2016, 17:1–9
2 Molecular physiology
Box 1 The beetle elytron. Primitive insects have two pairs of membranous flight wings, but during the evolution of the beetle lineage, the forewings lost their flight function and became modified as hard, rigid covers called ‘elytra’ [59–61], which is an example of appendage diversification of wings that have been diverted from their primary flight function and have evolved as a cuticular expansion [62]. This transformation is manifested by a greatly thickened and tanned elytral dorsal cuticle secreted by the forewing epidermis, and shares with the rigid body wall cuticle the same genetic cascades and tanning pathways in T. castaneum described above [6,47,63]. The elytron is composed of dorsal and ventral layers of epidermal cells that secrete a thick upper and a thin lower cuticular lamination, respectively [21,64,65]. The former is highly tanned and exhibits an ultrastructure very similar to that of other pigmented rigid cuticles in different body regions of adult beetles, whereas the latter is less pigmented and remains thin and membranous [21]. In addition, there is a space between these two layers, which is filled with hemolymph and a large number of supporting pillar-like fibrous structures called ‘trabeculae’ that generally have a mechanical function separating and supporting the dorsal and ventral cuticular layers of the elytron [50,66].
Elytron
Hindwing
DC, dorsal elytral cuticle: VC, ventral elytral cuticle: T, trabecula. Scale bar = 10 μm Current Opinion in Insect Science
N-acetyldopamine (NADA) or N-b-alanylation to N-balanyldopamine (NBAD), oxidation of NADA and NBAD to NADA-quinone and NBAD-quinone and their polymerization to form the corresponding pigments. It should be noted that these quinone metabolites produced by laccase 2-mediated oxidation reactions also undergo cross-linking reactions with side chains of CPs such as histidyl residues for cuticle sclerotization [7]. N-acylation of the catecholamines reduces the rate of intramolecular cyclization of their quinone derivatives such that protein cross-linking reactions via the quinones become more prevalent.
Beetle cuticle structural coloration Many beetle species including members of the families Scarabaeidae (scarab beetles) and Buprestidae (jewel beetles) have caught our attention because of their splendid metallic luster and iridescent body colors. Many of these beautiful colorations and some observed in several lepidopteran species [8–10] are actually ‘structural colors’ caused by a specific arrangement of cuticular structure(s) that differentially reflects light waves causing light interference. The structural coloration in beetles is mainly due to three iridescence mechanisms including multilayer reflectors, three-dimensional photonic crystals and diffraction gratings [11,12]. The multilayer reflector, which Current Opinion in Insect Science 2016, 17:1–9
is the most common and extensively studied mechanism, is composed of alternating highly electron-dense and electron-lucent layers. Depending on the number of layers and also the thickness and distance between successive layers, different optical colors are produced by interference of light waves. Recent studies have revealed that these reflectors can be found at different regions in beetle cuticles. For example, reflectors are found in the outer region of the cuticle (epicuticle and/or boundary between epicuticle and procuticle) of the jewel beetle, Chrysochroa fulgidissima [13], the aquatic leaf beetle, Plateumaris sericea [14] and the bronzed tiger beetle, Cicindela repanda [11], whereas they are localized in inner layers of the chitin-protein rich procuticle in many scarab beetles such as the rose chafer, Cetonia aurata [15]. Structural color, in addition, can sometimes be caused by a reversible change due to the environment. The yellow-greenish color of elytra of the Hercules beetle, Dynastes hercules, changes to black passively under high humidity conditions, and returns to a yellow-greenish color under dry conditions [16]. The yellow-greenish coloration is caused by a porous three-dimensional sponge-like structure located about 3 mm below the cuticular surface. The air in the holes of this structure are substituted for by water under high humidity, resulting in www.sciencedirect.com
Cuticle formation and pigmentation in beetles Noh et al.
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Box 2 Proposed metabolites (in black), enzymes/genes (in red) and structural proteins (CPs) in tyrosine-mediated cuticle pigmentation and sclerotization. Several studies have indicated the functional importance of the following metabolites and genes/enzymes/structural proteins in insect cuticle tanning reactions: tyrosine hydroxylase (TH) converts tyrosine to DOPA [67–73]; DOPA decarboxylase (DDC) converts DOPA to dopamine [68,70–73]; dopachrome conversion enzyme (DCE), which is encoded by some of the yellow family genes, appears to accelerate the conversion of dopachrome to DHI [73–78]; arylalkylamine N-acetyltransferase (aaNAT) converts dopamine to NADA [73,79–81]; aspartate 1-decarboxylase (ADC, Black) decarboxylates aspartic acid to b-alanine [82,83] for production of NBAD from dopamine catalyzed by NBAD synthase (Ebony) [70,71,74,75,77]; and laccase 2 catalyzes the catechol oxidation reactions in the tanning pathway [84–86]. A brief summary of the loss of function phenotypes produced by double-stranded RNA (dsRNA)-mediated gene silencing (RNAi) for those genes in the red flour beetle, T. castaneum, is as follows: TcTH: Injection of dsRNA for TcTH diminished the brown pigment in pupal cuticle on the abdominal segments, urogomphi, bristles and gin traps as well as in adult cuticle on the mandibles and legs, both of which were visible underneath the pupal cuticle, as well as the dark pigment present in the hindwings. The cuticle of hypomorphic dsTcTH-treated adults is pale [4]. TcDDC: Although the level of DOPA increases approximately 5-fold in the dsTcDDC-treated pharate adults, the initial cuticle pigmentation of the resulting adults is substantially delayed, suggesting that, unlike dopamine, DOPA is not well utilized for DOPA quinone-associated melanin synthesis, probably because DOPA is a poor substrate for laccase 2 [3]. The body color of the TcDDC-deficient mature adults is slightly darker than that of control animals. This phenotype may be due to a small amount of DOPA melanin accumulation relative to NBAD-derived and/or NADA-derived pigments in the adult cuticle. Tcebony and TcADC: Both genes are critical for NBAD synthesis in the cuticle tanning pathway. RNAi for Tcebony [63] and TcADC [3] caused a dark pigmented body color probably due to abnormally high levels of dopamine accumulated, which are used for dopamine quinone melanin production at the expense of NBAD quinone-mediated cross-linking and pigmentation during tanning. TcLac 2: Laccase 2 oxidizes catechols and catecholamines to their corresponding quinones/quinone methides, which are highly reactive crosslinking agents and indispensable to cuticle pigmentation and sclelotization. RNAi for laccase 2 (TcLac2) resulted in a decrease in pigmentation and hardness in larval, pupal and adult cuticles [87]. TcY-y and TcY-e: Yellow-y (Y-y) protein may act in the melanin synthetic pathway downstream from dopa and/or dopamine, although the precise function of the gene remains unclear [75]. In the presence of DmY-y in Drosophila, dopamine quinone polymerizes to form black melanin [88]. Mutation of the Y-y gene is responsible for the light brown/yellowish body color mutant of D. melanogaster and B. mori [75,77,78]. However, injection of dsTcY-y had no effect in Tribolium larvae, pupae and adults on body pigmentation except for black pigmentation in the pterostigma of the hindwing [6], suggesting that TcY-y is not critical for T. castaneum body wall cuticle pigmentation. Yellow-e (TcY-e)-depleted Tribolium adults died shortly after eclosion due to dehydration, and the lethality was prevented by high humidity [5]. The body color of the high humidity-rescued adults was significantly darker than that of control adults, suggesting that TcY-e plays a role not only in body pigmentation but also in cuticle waterproofing in T. castaneum adults.
Dopa-melanin
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5,6-dihydroxyindole-2carboxylic acid
5,6-dihydroxyindole
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Current Opinion in Insect Science 2016, 17:1–9
4 Molecular physiology
weak or no light backscattering, leaving a dark/black color that is likely produced by melanin-like pigments. This phenomenon is referred to as a diffractive hygrochromic effect.
Beetle cuticle morphology and structure Insect cuticle is composed of several morphologically and functionally distinct layers such as the envelope, epicuticle, exocuticle and endocuticle. The last two layers comprise the procuticle, which consists of a variable number of chitin-protein laminae arranged parallel to the apical plasma membrane of the epidermal cells [17]. The exo-cuticular and endo-cuticular layers are generally formed before and after molting, respectively. Scanning (SEM) and transmission electron microscopic (TEM) analyses of rigid elytral cuticle of about 40 species of beetles revealed its unique ultrastructural architecture [18,19]. Similarly, the rigid cuticle (e.g. thoracic-cuticle, leg-cuticle and elytral dorsal-cuticle) of the red flour beetle, Tribolium castaneum, is composed of the envelope, epicuticle, exocuticle, mesocuticle and endocuticle (Figure 1). In addition to the numerous horizontal laminae, there is a large number of vertical columnar structures in the dorsal elytral exocuticle, which extend
directly from the ‘apical plasma membrane protrusions’ (APMP) of the underlying epidermal cells, penetrate the horizontal laminae and reach all the way to the epicuticle. These vertical ‘pore canals’ contain chitin fibers denoted as ‘pore canal fibers’ (PCF) [20,21]. A similar ultrastructure containing PCFs has also been observed after removal of minerals and most proteins, also in the exoskeletons of crustaceans such as Homarus americanus (American lobster), Callinectes sapidus (Atlantic blue crab) and Tylos europaeus (sand-burrowing isopod) [22–24]. Roer et al. [25] suggested that the pore canals with PCFs are involved in the post-ecdysial transport of metabolites and macromolecules in the hard cuticles of hexapods and also in post-ecdysial mineralization in decapods. Further study, however, is required to identify what kind of molecule(s), if any, is transported through the pore canal/PCF during maturation of the hard cuticle. In the Japanese beetle, Popillia japonica, horizontal chitin-protein laminae in both the mesocuticle and exocuticle are helicoidally aligned, but those laminae are less tightly stacked in the former compared to those in the latter [26]. A similar less compact laminar arrangement in the mesocuticle is evident in the T. castaneum adult
Figure 1
EV EP
EXO
PCF MESO
ENDO PC
EC Current Opinion in Insect Science
Schematic diagram of the ultrastructure of rigid adult cuticle of a beetle. Adult rigid cuticle is composed of distinct layers including the envelope, epicuticle, exocuticle, mesocuticle and endocuticle. There are a number of horizontal chitinous laminae and numerous pore canals running transverse to the laminae in the exocuticle [21]. The pore canals extend from the apical plasma membrane to the epicuticlar region and contain a core of pore canal fibers. TEM image is of the dorsal elytral cuticle of 10 d-old T. castaneum adult. Scale bar = 2 mm. EV, envelope; EP, epicuticle; EXO, exocuticle; MESO, mesocuticle; ENDO, endocuticle; PC pore canal; PCF, pore canal fiber; EC, epidermal cell. Current Opinion in Insect Science 2016, 17:1–9
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Cuticle formation and pigmentation in beetles Noh et al.
exoskeleton (Figure 1 and see Figure 2 in Noh et al. [21] for details of tightly arranged laminae in the exocuticle). The innermost endocuticular layer (also denoted ‘macrofiber’ or ‘pseudo-orthogonal’ layer) is composed of several thick chitin-protein laminae, and constitutes a major portion of a beetles’ cuticle [18]. In T. castaneum, for example, this layer together with the mesocuticle accounts for about half of the total thickness of the rigid cuticle (Figure 1). It should be noted that laminae of endocuticle appear to be rotated at some angle because a number of the vertically oriented narrow pore canals are twisted and traverse discontinuously throughout the layer. Each lamina exhibits a different degree of electron density [18]. A similar laminar assembly in the endocuticle layer has been observed in adult cuticles from P. japonica, Oryctes rhinoceros (Asiatic rhinoceros beetle) and several weevils such as Anthonomus grandis, Trigonopterus nasutus, Sitophilus granarius and Cryptorhynchus lapathi [22,27,28]. In those substructures, successive unidirectionally oriented thick layers stack upon one another orthogonally with thin intervening transitional layers. Taken together, a beetle’s rigid cuticle exhibits a unique architecture consisting of complex ultrastructural layers. Because such ultrastructure has not been observed in the relatively soft and flexible cuticles of the larval body wall, adult dorsal abdomen and hindwing of T. castaneum [21] as well as not in the embryonic cuticle of the fruit fly, Drosophila melanogaster [17], this type of arrangement of sub layers with different architectures appears to contribute to the formation of rigid areas of the exoskeleton including the elytra of beetles.
Structural cuticle proteins (CPs) help to organize the procuticle CPs play a major role in determining the diverse physical properties of the cuticle, which vary at different developmental stages and in different regions of insect’s body as a result of interactions among themselves and with chitin [29]. Recent studies involving analyses of fully sequenced genomes revealed the presence of large numbers of genes encoding CPs in all insect species [30–35]. Insect CPs have been classified into thirteen distinct families based on the presence of unique amino acid sequence motifs [34,36,37]. The largest cuticular protein family is the CPR family whose members contain a conserved amino acid sequence known as the Rebers & Riddiford (R&R) consensus motif [38]. The R&R motif contains a chitin_bind_4 domain (PF00379 in the Pfam database, http://pfam.xfam.org), which may facilitate noncovalent interactions between chitin fibers and the proteinaceous matrix [35,39–41]. CPR proteins are further divided into subfamilies, two major, RR-1 and RR-2, and one minor and not yet well-defined, RR-3 [42,43]. The RR-1 and RR-2 families of proteins are found, with some exceptions, in soft/flexible and hard/rigid cuticles, respectively [37,43]. www.sciencedirect.com
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It is generally accepted that the degree of cross-linking of some CPs together with dehydration are critical for determining the nature of the cuticle with appropriate mechanical properties such as rigidity or flexibility. Several microarray and proteomics studies have revealed distinct expression patterns of CPs at different developmental stages or in different body regions [25,31, 33,35,44]. However, the functional significance and precise locations of CPs within a cuticle are still not well established. In the malaria mosquito, Anopheles gambiae, AgamCPF3 protein is localized in the adult’s exocuticle, while AgamCPLCG3/4 proteins are restricted to the endocuticle [45]. In the silkworm, Bombyx mori, mutation of the BmorCPR2 gene is responsible for the stony mutant. In this mutant, a dysfunctional BmorCPR2 protein (RR1) causes an abnormal distribution of internodes and intersegmental folds, and reductions in both chitin content and tensile/stretch properties of the larval cuticle [46]. In the beetle T. castaneum, functional studies using immunohistochemistry, electron microscopic immunocytochemistry and RNAi of four major CPs in the adult cuticle, TcCPR27, TcCPR18, TcCPR4 and TcCP30, have provided some glimpses into the roles of CPs in organizing the cuticle and shaping its properties [20,47,48]. TcCPR27 and TcCPR18, which are the most abundant cuticular proteins in hard cuticles, are members of the CPR family containing an RR-2 motif, and are localized in both the chitinous horizontal laminae and vertical pore canals of rigid adult cuticle [21]. RNAi for TcCPR27 or TcCPR18 genes caused a disorganization of the laminar architecture and amorphous pore canal fibers (PCFs), which resulted in short, wrinkled and weakened elytra and compromised their structural integrity [21,47]. TcCPR4, which contains an RR-1 motif, is specifically localized with the PCFs in the vicinity of the APMPs, but it is absent or much less abundant in the horizontal laminae of the procuticle [20]. Interestingly, in the rigid cuticle of TcCPR27-deficient adults, TcCPR4 is mislocalized and distributed over the entire exocuticle including the horizontal laminae, suggesting that the presence of TcCPR27 protein is critical for localization of the TcCPR4 protein in the pore canals. In the TcCPR4-deficient cuticle, abnormal pore canals with amorphous PCFs in their lumen are evident, indicating that TcCPR4 is important for determining the morphology and ultrastructure of the PCFs and pore canals in rigid cuticle. Like TcCPR27, TcCPR18 and TcCPR4, TcCP30 is abundant in rigid cuticles (e.g. dorsal elytral cuticle, pronotum and leg), but not in soft and flexible cuticles (e.g. hindwing and dorsal abdominal cuticle) of the adult [48]. However, unlike the former three TcCPRs, TcCP30 has a low sequence complexity with a unique amino acid composition and is not a member of any known CP family. Loss of function of TcCP30 by RNAi led to 70% of a lethal pupal-adult molting defect Current Opinion in Insect Science 2016, 17:1–9
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phenotype. The resulting adults that did undergo eclosion, in addition, exhibited wrinkled and separated elytra and improperly folding hindwings. Western blotting analysis revealed that TcCP30 becomes cross-linked to TcCPR27, TcCPR18 and itself (and probably other CPs), but not to TcCPR4, by the action of laccase 2 [48]. All of these results indicate that a unique localization and cross-linking of specific CPs are critical for maintaining the morphology and ultrastructure of the beetle exoskeleton (Figure 2) as well as for their development and survival. What other CP(s) comprise the exocuticular, mesocuticular and endocuticular layers remain an area worthy of future investigation.
Future prospects and concluding remarks A number of studies have been conducted to understand and utilize the structural properties that insects possess [49]. One of the most active research areas is biomimetic science, which involves the development of novel cuticleinspired materials with unique physical properties similar to those of the insect exoskeleton for use in biomedical or other technological devices. For example, the highly
modified and tanned forewing or elytron of beetles (Box 1) is an enriched source of cuticle-forming epidermis, easily separable from other tissues, making it an ideal tissue for study of biochemical, ultrastructural and mechanical properties of rigid cuticle and as a biomimetic model and its application [12,50,51,52,53]. Unlike other tanned cuticles in different body regions of adult beetles, the elytron is composed of dual cuticular layers connected by a large number of pillar-like fibrous structures called trabeculae. The unique structure of the elytron has been applied as a model to develop novel integrated honeycomb structures that are lightweight with high mechanical strength [12,54]. The color and metallic sheen of beetle elytra are also being studied in a biomimetic search for material-structuring principles causing light, thermal and electrical interferences [55]. Their brilliant metallic appearance originates from structures in organic materials that are both electrically and thermally insulating. Thus, there is an industrial effort to develop insect cuticleinspired products with attractive metallic surfaces that do not feel so cold as their metallic counterparts and do not present an electrical shock hazard.
Figure 2
(a)
(b)
EV EP
Pr
ote
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ot
ei n
in
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N
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Proposed cross-linking network of cuticular proteins, chitin and catechols in rigid cuticle of a beetle. (a) Histidine residue(s) of CPs likely participate in cross-linking reactions through the quinone- and/or quinone methide intermediates derived from NADA and/or NBAD during cuticle maturation [7]. Addition adducts at either the 2-carbon and 6-carbon of the catechol ring or the b-carbon of the histidyl side chain with an imidazole nitrogen and/or the amino group of glucosamine are proposed to occur [56–58]. R COCH3 and COCH2CH2NH2 are NADA and NBAD, respectively. (b)Major CPs in rigid cuticle of T. castaneum. TcCPR27, TcCPR18 and TcCP30 are localized in both laminae and pore canal fibers, whereas TcCPR4 is predominantly localized in pore canal fibers [20,21,48]. TcCP30 undergoes cross-linking with TcCPR27 and TcCPR18, but not to TcCPR4 in rigid cuticle [48]. Abbreviations are listed in the legend of Figure 1. Current Opinion in Insect Science 2016, 17:1–9
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Cuticle formation and pigmentation in beetles Noh et al.
Current research is also focused on identifying the role(s) of individual CPs by following their expression profiles, localization and alterations in phenotypes as well as insect viability following depletion of specific cuticular components. Additional experiments are needed to determine the precise timing of appearance and location of individual components, their transport into and throughout the cuticle, and how they interact with one another to shape the overall architecture and properties of the exoskeleton. Why there is a need for such a large assortment of families of CPs and multiple members within each family and how they influence the properties of diverse cuticular structures will be the focus of future studies.
Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A2A01006614), and Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2015R1A6A3A04060323) to MYN.
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21. Noh MY, Kramer KJ, Muthukrishnan S, Kanost MR, Beeman RW, Arakane Y: Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle of Tribolium castaneum. Insect Biochem Mol Biol 2014, 53:22-29. This article describes the localization and functional importance of two major cuticle proteins in rigid cuticle morphology and ultrastructure of T. castaneum adults.
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31. Dittmer NT, Hiromasa Y, Tomich JM, Lu N, Beeman RW, Kramer KJ, Kanost MR: Proteomic and transcriptomic analyses of rigid and membranous cuticles and epidermis from the elytra and hindwings of the red flour beetle, Tribolium castaneum. J Proteome Res 2012, 11:269-278.
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51. Chen J, Xie J, Wu Z, Elbashiry EM, Lu Y: Review of beetle forewing structures and their biomimetic applications in China: (I) On the structural colors and the vertical and horizontal cross-sectional structures. Mater Sci Eng C Mater Biol Appl 2015, 55:605-619. This review describes the morphology and ultrastructure of the elytral cuticle from several beetle species, which contribute to structural coloration and mechanical properties. 52. Dai ZD, Yang ZX: Macro-/micro-structures of elytra, mechanical properties of the biomaterial and the coupling strength between elytra in beetles. J Biomim Eng 2010, 7:6-12. 53. Lomakin J, Huber PA, Eichler C, Arakane Y, Kramer KJ, Beeman RW, Kanost MR, Gehrke SH: Mechanical properties of the beetle elytron, a biological composite material. Biomacromolecules 2011, 12:321-335. 54. Chen J, Xie J, Zhu H, Guan S, Wu G, Noori MN, Guo S: Integrated honeycomb structure of a beetle forewing and its imitation. Mater Sci Eng C 2012, 32:613-618. 55. Lenau T, Barfoed M: Colours and metallic sheen in beetle shells — a biomimetic search for material structuring principles causing light interference. Adv Eng Mater 2008, 10:299-314. 56. Xu RD, Huang X, Hopkins TL, Kramer KJ: Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochem Mol Biol 1997, 27:101-108. 57. Huang X, Xu RD, Hawley MD, Kramer KJ: Model insect cuticle sclerotization: reactions of catecholamine quinones with the nitrogen-centered nucleophiles imidazole and Nacetylhistidine. Bioorg Chem 1997, 25:179-202. 58. Xu R, Huang X, Morgan TD, Prakash O, Kramer KJ, Hawley MD: Characterization of products from the reactions of Nacetyldopamine quinone with N-acetylhistidine. Arch Biochem Biophys 1996, 329:56-64. www.sciencedirect.com
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59. Tomoyasu Y, Wheeler SR, Denell RE: Ultrabithorax is required for membranous wing identity in the beetle Tribolium castaneum. Nature 2005, 433:643-647. 60. Hunt T, Bergsten J, Levkanicova Z, Papadopoulou A, John OS, Wild R, Hammond PM, Ahrens D, Balke M, Caterino MS et al.: A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation. Science 2007, 318:1913-1916. 61. Grimaldi D, Engel MS: Evolution of the Insects. New York: Cambridge University Press; 2005. 62. Prud’homme B, Minervino C, Hocine M, Cande JD, Aouane A, Dufour HD, Kassner VA, Gompel N: Body plan innovation in treehoppers through the evolution of an extra wing-like appendage. Nature 2011, 473:83-86. 63. Tomoyasu Y, Arakane Y, Kramer KJ, Denell RE: Repeated co options of exoskeleton formation during wing-to-elytron evolution in beetles. Curr Biol 2009, 19:2057-2065. This paper demonstrates that a co-opted pathway downstream of the conserved basic wing gene network rather than changes in the gene network itself is responsible for transformation of the membranous forewings into thickened and hardend non-flight covers (elytra) in a process called ‘exoskeletalization’. 64. Chen J, Dai G, Xu Y, Iwamoto M: Optimal composite structures in the forewings of beetles. Compos Struct 2007, 81:432-437. 65. Chen J, Ni QQ, Xu Y, Iwamoto M: Lightweight composite structures in the forewings of beetles. Compos Struct 2007, 79:331-337. 66. Ni QQ, Chen JX, Iwamoto M, Kurashiki K, Saito K: Interlaminar reinforcement mechanism in a beetle fore-wing. JSME Int J CMech Syst 2001, 44:1111-1116. 67. Liu C, Yamamoto K, Cheng TC, Kadono-Okuda K, Narukawa J, Liu SP, Han Y, Futahashi R, Kidokoro K, Noda H et al.: Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, Bombyx mori. Proc Natl Acad Sci U S A 2010, 107:12980-12985. 68. Liu J, Lemonds TR, Popadic A: The genetic control of aposematic black pigmentation in hemimetabolous insects: insights from Oncopeltus fasciatus. Evol Dev 2014, 16:270-277. 69. Liu S, Wang M, Li X: Overexpression of Tyrosine hydroxylase and Dopa decarboxylase associated with pupal melanization in Spodoptera exigua. Sci Rep 2015, 5:11273. 70. Futahashi R, Fujiwara H: Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, Papilio xuthus. Dev Genes Evol 2005, 215:519-529. 71. True JR, Edwards KA, Yamamoto D, Carroll SB: Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns. Curr Biol 1999, 9:1382-1391. 72. Ninomiya Y, Tanaka K, Hayakawa Y: Mechanisms of black and white stripe pattern formation in the cuticles of insect larvae. J Insect Physiol 2006, 52:638-645.
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butterfly, Papilio xuthus. Insect Biochem Mol Biol 2007, 37:855-864. 75. Futahashi R, Sato J, Meng Y, Okamoto S, Daimon T, Yamamoto K, Suetsugu Y, Narukawa J, Takahashi H, Banno Y et al.: yellow and ebony are the responsible genes for the larval color mutants of the silkworm Bombyx mori. Genetics 2008, 180:1995-2005. 76. Ito K, Katsuma S, Yamamoto K, Kadono-Okuda K, Mita K, Shimada T: Yellow-e determines the color pattern of larval head and tail spots of the silkworm Bombyx mori. J Biol Chem 2010, 285:5624-5629. 77. Wittkopp PJ, True JR, Carroll SB: Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns. Development 2002, 129:1849-1858. 78. Wittkopp PJ, Vaccaro K, Carroll SB: Evolution of yellow gene regulation and pigmentation in Drosophila. Curr Biol 2002, 12:1547-1556. 79. Dai FY, Qiao L, Tong XL, Cao C, Chen P, Chen J, Lu C, Xiang ZH: Mutations of an arylalkylamine-N-acetyltransferase, BmiAANAT, are responsible for silkworm melanism mutant. J Biol Chem 2010, 285:19553-19560. 80. Qiao L, Li Y, Xiong G, Liu X, He S, Tong X, Wu S, Hu H, Wang R, Hu H et al.: Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant. PLoS ONE 2012, 7:e42968. 81. Zhan S, Guo Q, Li M, Li M, Li J, Miao X, Huang Y: Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation. Development 2010, 137:4083-4090. 82. Dai F, Qiao L, Cao C, Liu X, Tong X, He S, Hu H, Zhang L, Wu S, Tan D et al.: Aspartate decarboxylase is required for a normal pupa pigmentation pattern in the silkworm, Bombyx mori. Sci Rep 2015, 5:10885. 83. Phillips AM, Smart R, Strauss R, Brembs B, Kelly LE: The Drosophila black enigma: the molecular and behavioural characterization of the black1 mutant allele. Gene 2005, 351:131-142. 84. Futahashi R, Banno Y, Fujiwara H: Caterpillar color patterns are determined by a two-phase melanin gene prepatterning process: new evidence from tan and laccase2. Evol Dev 2010, 12:157-167. 85. Futahashi R, Tanaka K, Matsuura Y, Tanahashi M, Kikuchi Y, Fukatsu T: Laccase2 is required for cuticular pigmentation in stinkbugs. Insect Biochem Mol Biol 2011, 41:191-196. 86. Elias-Neto M, Soares MP, Simoes ZL, Hartfelder K, Bitondi MM: Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae). Insect Biochem Mol Biol 2010, 40:241-251.
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Current Opinion in Insect Science 2016, 17:1–9
ScienceDirect Cuticle formation and pigmentation in beetles Mi Young Noh1, Subbaratnam Muthukrishnan2, Karl J Kramer2 and Yasuyuki Arakane1 Adult beetles (Coleoptera) are covered primarily by a hard exoskeleton or cuticle. For example, the beetle elytron is a cuticle-rich highly modified forewing structure that shields the underlying hindwing and dorsal body surface from a variety of harmful environmental factors by acting as an armor plate. The elytron comes in a variety of colors and shapes depending on the coleopteran species. As in many other insect species, the cuticular tanning pathway begins with tyrosine and is responsible for production of a variety of melanin-like and other types of pigments. Tanning metabolism involves quinones and quinone methides, which also act as protein cross-linking agents for cuticle sclerotization. Electron microscopic analyses of rigid cuticles of the red flour beetle, Tribolium castaneum, have revealed not only numerous horizontal chitin-protein laminae but also vertically oriented columnar structures called pore canal fibers. This structural architecture together with tyrosine metabolism for cuticle tanning is likely to contribute to the rigidity and coloration of the beetle exoskeleton. Addresses 1 Department of Applied Biology, Chonnam National University, Gwangju 61186, Republic of Korea 2 Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, United States Corresponding author: Arakane, Yasuyuki ([email protected])
Current Opinion in Insect Science 2016, 17:1–9 This review comes from a themed issue on Molecular physiology Edited by Takema Fukatsu and Ryo Futahashi For a complete overview see the Issue and the Editorial Available online 12th May 2016 http://dx.doi.org/10.1016/j.cois.2016.05.004 2214-5745/# 2016 Elsevier Inc. All rights reserved.
and skeleton, protecting insects from environmental stresses and mechanical damage. The Coleoptera is the largest insect order, and its cuticle including that of the elytron (Box 1), which is a highly modified and tanned (sclerotized and pigmented) forewing, has been an attractive material for study not only because of its metallic and lustrous coloration but also due to its mechanical properties as a rigid and lightweight tissue. Here, we review the functional importance and the diversity of the genes involved in the tyrosine-derived cuticle tanning pathway, and the morphology and ultrastructure of rigid types of beetle cuticle. The functional importance, unique localization, and cross-linking of specific CPs in the formation of the exoskeleton are also discussed.
Beetle cuticle pigmentation Several kinds of chemical pigments such as melanins, pterins, ommochromes, antraquinones, aphins, tertapyrroles, carotenoids and flavonoids/anthocyanins contribute to a variety of insect colors [1]. However, there have been only a few studies focusing on the identification, biosynthesis, genetic regulation and function of these pigments in beetles. For example, carotenoid content is responsible for the variable orange-to-red coloration of elytra of the Asian ladybird beetle, Harmonia axyridis, and this red hue appears to be aposematically correlated with the content of their defensive alkaloid molecules [2]. Like other insect species, melanin-like and quinonoid pigments produced by tyrosine metabolism play a major role in the darkening of beetle cuticle [3,4,5,6]. Tyrosine metabolism is also critical for cuticle hardening (sclerotization). Cuticle tanning involving tyrosine metabolism has been a study of major emphasis in our laboratories.
Tyrosine-mediated cuticle pigmentation and sclerotization Introduction Insects, belonging to the most diverse and successful animal phylum Arthropoda, have developed and acquired superior adaptability to the natural environment, excellent communication systems, and optimized body designs and functions throughout their evolution. In addition to these features, the exoskeleton or cuticle gives the insects excellent capabilities that help to account for their evolutionary success. The insect cuticle is a remarkable biomaterial/biomass primarily formed from structural cuticle proteins (CPs) and the linear polysaccharide, chitin. This strong extracellular material serves both as a skin www.sciencedirect.com
Cuticle tanning (pigmentation and sclerotization) is a complex process, which includes hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and decarboxylation of DOPA to dopamine (Box 2). Additional steps for melanization include oxidation of DOPA and dopamine to dopa-quinone and dopamine-quinone, conversion of these quinones to dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), via intramolecular cyclization, oxidation of DHI and DHICA to DHI-chrome and DHICA-chrome (melanochromes) and then polymerization of the melanochromes to form melanin-like pigments. Pigmentation involving N-acylated quinones requires N-acetylation of dopamine to Current Opinion in Insect Science 2016, 17:1–9
2 Molecular physiology
Box 1 The beetle elytron. Primitive insects have two pairs of membranous flight wings, but during the evolution of the beetle lineage, the forewings lost their flight function and became modified as hard, rigid covers called ‘elytra’ [59–61], which is an example of appendage diversification of wings that have been diverted from their primary flight function and have evolved as a cuticular expansion [62]. This transformation is manifested by a greatly thickened and tanned elytral dorsal cuticle secreted by the forewing epidermis, and shares with the rigid body wall cuticle the same genetic cascades and tanning pathways in T. castaneum described above [6,47,63]. The elytron is composed of dorsal and ventral layers of epidermal cells that secrete a thick upper and a thin lower cuticular lamination, respectively [21,64,65]. The former is highly tanned and exhibits an ultrastructure very similar to that of other pigmented rigid cuticles in different body regions of adult beetles, whereas the latter is less pigmented and remains thin and membranous [21]. In addition, there is a space between these two layers, which is filled with hemolymph and a large number of supporting pillar-like fibrous structures called ‘trabeculae’ that generally have a mechanical function separating and supporting the dorsal and ventral cuticular layers of the elytron [50,66].
Elytron
Hindwing
DC, dorsal elytral cuticle: VC, ventral elytral cuticle: T, trabecula. Scale bar = 10 μm Current Opinion in Insect Science
N-acetyldopamine (NADA) or N-b-alanylation to N-balanyldopamine (NBAD), oxidation of NADA and NBAD to NADA-quinone and NBAD-quinone and their polymerization to form the corresponding pigments. It should be noted that these quinone metabolites produced by laccase 2-mediated oxidation reactions also undergo cross-linking reactions with side chains of CPs such as histidyl residues for cuticle sclerotization [7]. N-acylation of the catecholamines reduces the rate of intramolecular cyclization of their quinone derivatives such that protein cross-linking reactions via the quinones become more prevalent.
Beetle cuticle structural coloration Many beetle species including members of the families Scarabaeidae (scarab beetles) and Buprestidae (jewel beetles) have caught our attention because of their splendid metallic luster and iridescent body colors. Many of these beautiful colorations and some observed in several lepidopteran species [8–10] are actually ‘structural colors’ caused by a specific arrangement of cuticular structure(s) that differentially reflects light waves causing light interference. The structural coloration in beetles is mainly due to three iridescence mechanisms including multilayer reflectors, three-dimensional photonic crystals and diffraction gratings [11,12]. The multilayer reflector, which Current Opinion in Insect Science 2016, 17:1–9
is the most common and extensively studied mechanism, is composed of alternating highly electron-dense and electron-lucent layers. Depending on the number of layers and also the thickness and distance between successive layers, different optical colors are produced by interference of light waves. Recent studies have revealed that these reflectors can be found at different regions in beetle cuticles. For example, reflectors are found in the outer region of the cuticle (epicuticle and/or boundary between epicuticle and procuticle) of the jewel beetle, Chrysochroa fulgidissima [13], the aquatic leaf beetle, Plateumaris sericea [14] and the bronzed tiger beetle, Cicindela repanda [11], whereas they are localized in inner layers of the chitin-protein rich procuticle in many scarab beetles such as the rose chafer, Cetonia aurata [15]. Structural color, in addition, can sometimes be caused by a reversible change due to the environment. The yellow-greenish color of elytra of the Hercules beetle, Dynastes hercules, changes to black passively under high humidity conditions, and returns to a yellow-greenish color under dry conditions [16]. The yellow-greenish coloration is caused by a porous three-dimensional sponge-like structure located about 3 mm below the cuticular surface. The air in the holes of this structure are substituted for by water under high humidity, resulting in www.sciencedirect.com
Cuticle formation and pigmentation in beetles Noh et al.
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Box 2 Proposed metabolites (in black), enzymes/genes (in red) and structural proteins (CPs) in tyrosine-mediated cuticle pigmentation and sclerotization. Several studies have indicated the functional importance of the following metabolites and genes/enzymes/structural proteins in insect cuticle tanning reactions: tyrosine hydroxylase (TH) converts tyrosine to DOPA [67–73]; DOPA decarboxylase (DDC) converts DOPA to dopamine [68,70–73]; dopachrome conversion enzyme (DCE), which is encoded by some of the yellow family genes, appears to accelerate the conversion of dopachrome to DHI [73–78]; arylalkylamine N-acetyltransferase (aaNAT) converts dopamine to NADA [73,79–81]; aspartate 1-decarboxylase (ADC, Black) decarboxylates aspartic acid to b-alanine [82,83] for production of NBAD from dopamine catalyzed by NBAD synthase (Ebony) [70,71,74,75,77]; and laccase 2 catalyzes the catechol oxidation reactions in the tanning pathway [84–86]. A brief summary of the loss of function phenotypes produced by double-stranded RNA (dsRNA)-mediated gene silencing (RNAi) for those genes in the red flour beetle, T. castaneum, is as follows: TcTH: Injection of dsRNA for TcTH diminished the brown pigment in pupal cuticle on the abdominal segments, urogomphi, bristles and gin traps as well as in adult cuticle on the mandibles and legs, both of which were visible underneath the pupal cuticle, as well as the dark pigment present in the hindwings. The cuticle of hypomorphic dsTcTH-treated adults is pale [4]. TcDDC: Although the level of DOPA increases approximately 5-fold in the dsTcDDC-treated pharate adults, the initial cuticle pigmentation of the resulting adults is substantially delayed, suggesting that, unlike dopamine, DOPA is not well utilized for DOPA quinone-associated melanin synthesis, probably because DOPA is a poor substrate for laccase 2 [3]. The body color of the TcDDC-deficient mature adults is slightly darker than that of control animals. This phenotype may be due to a small amount of DOPA melanin accumulation relative to NBAD-derived and/or NADA-derived pigments in the adult cuticle. Tcebony and TcADC: Both genes are critical for NBAD synthesis in the cuticle tanning pathway. RNAi for Tcebony [63] and TcADC [3] caused a dark pigmented body color probably due to abnormally high levels of dopamine accumulated, which are used for dopamine quinone melanin production at the expense of NBAD quinone-mediated cross-linking and pigmentation during tanning. TcLac 2: Laccase 2 oxidizes catechols and catecholamines to their corresponding quinones/quinone methides, which are highly reactive crosslinking agents and indispensable to cuticle pigmentation and sclelotization. RNAi for laccase 2 (TcLac2) resulted in a decrease in pigmentation and hardness in larval, pupal and adult cuticles [87]. TcY-y and TcY-e: Yellow-y (Y-y) protein may act in the melanin synthetic pathway downstream from dopa and/or dopamine, although the precise function of the gene remains unclear [75]. In the presence of DmY-y in Drosophila, dopamine quinone polymerizes to form black melanin [88]. Mutation of the Y-y gene is responsible for the light brown/yellowish body color mutant of D. melanogaster and B. mori [75,77,78]. However, injection of dsTcY-y had no effect in Tribolium larvae, pupae and adults on body pigmentation except for black pigmentation in the pterostigma of the hindwing [6], suggesting that TcY-y is not critical for T. castaneum body wall cuticle pigmentation. Yellow-e (TcY-e)-depleted Tribolium adults died shortly after eclosion due to dehydration, and the lethality was prevented by high humidity [5]. The body color of the high humidity-rescued adults was significantly darker than that of control adults, suggesting that TcY-e plays a role not only in body pigmentation but also in cuticle waterproofing in T. castaneum adults.
Dopa-melanin
Dopamine-melanin
Melanochrome
Melanochrome
Lac2
Lac2
5,6-dihydroxyindole-2carboxylic acid
5,6-dihydroxyindole
DCE (yellow)
DCE (yellow)
Dopachrome
Dopaminechrome
Dopa-quinone
Dopaminequinone
NBAD-pigment
Cuticle
NADA-pigment
Sclerotization
CP
Lac2
Lac2
CP
(NADA-sclerotin)
(NBAD-sclerotin)
NADA-quinone
NBAD-quinone
Lac2
Lac2
Epidermal cell
NADA aaNAT
DOPA
DDC
Dopamine
NBAD NBADH (tan) NBAD synthase (ebony)
β-alanine
TH
ADC
Tyrosine
L-Aspartic acid Current Opinion in Insect Science
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Current Opinion in Insect Science 2016, 17:1–9
4 Molecular physiology
weak or no light backscattering, leaving a dark/black color that is likely produced by melanin-like pigments. This phenomenon is referred to as a diffractive hygrochromic effect.
Beetle cuticle morphology and structure Insect cuticle is composed of several morphologically and functionally distinct layers such as the envelope, epicuticle, exocuticle and endocuticle. The last two layers comprise the procuticle, which consists of a variable number of chitin-protein laminae arranged parallel to the apical plasma membrane of the epidermal cells [17]. The exo-cuticular and endo-cuticular layers are generally formed before and after molting, respectively. Scanning (SEM) and transmission electron microscopic (TEM) analyses of rigid elytral cuticle of about 40 species of beetles revealed its unique ultrastructural architecture [18,19]. Similarly, the rigid cuticle (e.g. thoracic-cuticle, leg-cuticle and elytral dorsal-cuticle) of the red flour beetle, Tribolium castaneum, is composed of the envelope, epicuticle, exocuticle, mesocuticle and endocuticle (Figure 1). In addition to the numerous horizontal laminae, there is a large number of vertical columnar structures in the dorsal elytral exocuticle, which extend
directly from the ‘apical plasma membrane protrusions’ (APMP) of the underlying epidermal cells, penetrate the horizontal laminae and reach all the way to the epicuticle. These vertical ‘pore canals’ contain chitin fibers denoted as ‘pore canal fibers’ (PCF) [20,21]. A similar ultrastructure containing PCFs has also been observed after removal of minerals and most proteins, also in the exoskeletons of crustaceans such as Homarus americanus (American lobster), Callinectes sapidus (Atlantic blue crab) and Tylos europaeus (sand-burrowing isopod) [22–24]. Roer et al. [25] suggested that the pore canals with PCFs are involved in the post-ecdysial transport of metabolites and macromolecules in the hard cuticles of hexapods and also in post-ecdysial mineralization in decapods. Further study, however, is required to identify what kind of molecule(s), if any, is transported through the pore canal/PCF during maturation of the hard cuticle. In the Japanese beetle, Popillia japonica, horizontal chitin-protein laminae in both the mesocuticle and exocuticle are helicoidally aligned, but those laminae are less tightly stacked in the former compared to those in the latter [26]. A similar less compact laminar arrangement in the mesocuticle is evident in the T. castaneum adult
Figure 1
EV EP
EXO
PCF MESO
ENDO PC
EC Current Opinion in Insect Science
Schematic diagram of the ultrastructure of rigid adult cuticle of a beetle. Adult rigid cuticle is composed of distinct layers including the envelope, epicuticle, exocuticle, mesocuticle and endocuticle. There are a number of horizontal chitinous laminae and numerous pore canals running transverse to the laminae in the exocuticle [21]. The pore canals extend from the apical plasma membrane to the epicuticlar region and contain a core of pore canal fibers. TEM image is of the dorsal elytral cuticle of 10 d-old T. castaneum adult. Scale bar = 2 mm. EV, envelope; EP, epicuticle; EXO, exocuticle; MESO, mesocuticle; ENDO, endocuticle; PC pore canal; PCF, pore canal fiber; EC, epidermal cell. Current Opinion in Insect Science 2016, 17:1–9
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Cuticle formation and pigmentation in beetles Noh et al.
exoskeleton (Figure 1 and see Figure 2 in Noh et al. [21] for details of tightly arranged laminae in the exocuticle). The innermost endocuticular layer (also denoted ‘macrofiber’ or ‘pseudo-orthogonal’ layer) is composed of several thick chitin-protein laminae, and constitutes a major portion of a beetles’ cuticle [18]. In T. castaneum, for example, this layer together with the mesocuticle accounts for about half of the total thickness of the rigid cuticle (Figure 1). It should be noted that laminae of endocuticle appear to be rotated at some angle because a number of the vertically oriented narrow pore canals are twisted and traverse discontinuously throughout the layer. Each lamina exhibits a different degree of electron density [18]. A similar laminar assembly in the endocuticle layer has been observed in adult cuticles from P. japonica, Oryctes rhinoceros (Asiatic rhinoceros beetle) and several weevils such as Anthonomus grandis, Trigonopterus nasutus, Sitophilus granarius and Cryptorhynchus lapathi [22,27,28]. In those substructures, successive unidirectionally oriented thick layers stack upon one another orthogonally with thin intervening transitional layers. Taken together, a beetle’s rigid cuticle exhibits a unique architecture consisting of complex ultrastructural layers. Because such ultrastructure has not been observed in the relatively soft and flexible cuticles of the larval body wall, adult dorsal abdomen and hindwing of T. castaneum [21] as well as not in the embryonic cuticle of the fruit fly, Drosophila melanogaster [17], this type of arrangement of sub layers with different architectures appears to contribute to the formation of rigid areas of the exoskeleton including the elytra of beetles.
Structural cuticle proteins (CPs) help to organize the procuticle CPs play a major role in determining the diverse physical properties of the cuticle, which vary at different developmental stages and in different regions of insect’s body as a result of interactions among themselves and with chitin [29]. Recent studies involving analyses of fully sequenced genomes revealed the presence of large numbers of genes encoding CPs in all insect species [30–35]. Insect CPs have been classified into thirteen distinct families based on the presence of unique amino acid sequence motifs [34,36,37]. The largest cuticular protein family is the CPR family whose members contain a conserved amino acid sequence known as the Rebers & Riddiford (R&R) consensus motif [38]. The R&R motif contains a chitin_bind_4 domain (PF00379 in the Pfam database, http://pfam.xfam.org), which may facilitate noncovalent interactions between chitin fibers and the proteinaceous matrix [35,39–41]. CPR proteins are further divided into subfamilies, two major, RR-1 and RR-2, and one minor and not yet well-defined, RR-3 [42,43]. The RR-1 and RR-2 families of proteins are found, with some exceptions, in soft/flexible and hard/rigid cuticles, respectively [37,43]. www.sciencedirect.com
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It is generally accepted that the degree of cross-linking of some CPs together with dehydration are critical for determining the nature of the cuticle with appropriate mechanical properties such as rigidity or flexibility. Several microarray and proteomics studies have revealed distinct expression patterns of CPs at different developmental stages or in different body regions [25,31, 33,35,44]. However, the functional significance and precise locations of CPs within a cuticle are still not well established. In the malaria mosquito, Anopheles gambiae, AgamCPF3 protein is localized in the adult’s exocuticle, while AgamCPLCG3/4 proteins are restricted to the endocuticle [45]. In the silkworm, Bombyx mori, mutation of the BmorCPR2 gene is responsible for the stony mutant. In this mutant, a dysfunctional BmorCPR2 protein (RR1) causes an abnormal distribution of internodes and intersegmental folds, and reductions in both chitin content and tensile/stretch properties of the larval cuticle [46]. In the beetle T. castaneum, functional studies using immunohistochemistry, electron microscopic immunocytochemistry and RNAi of four major CPs in the adult cuticle, TcCPR27, TcCPR18, TcCPR4 and TcCP30, have provided some glimpses into the roles of CPs in organizing the cuticle and shaping its properties [20,47,48]. TcCPR27 and TcCPR18, which are the most abundant cuticular proteins in hard cuticles, are members of the CPR family containing an RR-2 motif, and are localized in both the chitinous horizontal laminae and vertical pore canals of rigid adult cuticle [21]. RNAi for TcCPR27 or TcCPR18 genes caused a disorganization of the laminar architecture and amorphous pore canal fibers (PCFs), which resulted in short, wrinkled and weakened elytra and compromised their structural integrity [21,47]. TcCPR4, which contains an RR-1 motif, is specifically localized with the PCFs in the vicinity of the APMPs, but it is absent or much less abundant in the horizontal laminae of the procuticle [20]. Interestingly, in the rigid cuticle of TcCPR27-deficient adults, TcCPR4 is mislocalized and distributed over the entire exocuticle including the horizontal laminae, suggesting that the presence of TcCPR27 protein is critical for localization of the TcCPR4 protein in the pore canals. In the TcCPR4-deficient cuticle, abnormal pore canals with amorphous PCFs in their lumen are evident, indicating that TcCPR4 is important for determining the morphology and ultrastructure of the PCFs and pore canals in rigid cuticle. Like TcCPR27, TcCPR18 and TcCPR4, TcCP30 is abundant in rigid cuticles (e.g. dorsal elytral cuticle, pronotum and leg), but not in soft and flexible cuticles (e.g. hindwing and dorsal abdominal cuticle) of the adult [48]. However, unlike the former three TcCPRs, TcCP30 has a low sequence complexity with a unique amino acid composition and is not a member of any known CP family. Loss of function of TcCP30 by RNAi led to 70% of a lethal pupal-adult molting defect Current Opinion in Insect Science 2016, 17:1–9
6 Molecular physiology
phenotype. The resulting adults that did undergo eclosion, in addition, exhibited wrinkled and separated elytra and improperly folding hindwings. Western blotting analysis revealed that TcCP30 becomes cross-linked to TcCPR27, TcCPR18 and itself (and probably other CPs), but not to TcCPR4, by the action of laccase 2 [48]. All of these results indicate that a unique localization and cross-linking of specific CPs are critical for maintaining the morphology and ultrastructure of the beetle exoskeleton (Figure 2) as well as for their development and survival. What other CP(s) comprise the exocuticular, mesocuticular and endocuticular layers remain an area worthy of future investigation.
Future prospects and concluding remarks A number of studies have been conducted to understand and utilize the structural properties that insects possess [49]. One of the most active research areas is biomimetic science, which involves the development of novel cuticleinspired materials with unique physical properties similar to those of the insect exoskeleton for use in biomedical or other technological devices. For example, the highly
modified and tanned forewing or elytron of beetles (Box 1) is an enriched source of cuticle-forming epidermis, easily separable from other tissues, making it an ideal tissue for study of biochemical, ultrastructural and mechanical properties of rigid cuticle and as a biomimetic model and its application [12,50,51,52,53]. Unlike other tanned cuticles in different body regions of adult beetles, the elytron is composed of dual cuticular layers connected by a large number of pillar-like fibrous structures called trabeculae. The unique structure of the elytron has been applied as a model to develop novel integrated honeycomb structures that are lightweight with high mechanical strength [12,54]. The color and metallic sheen of beetle elytra are also being studied in a biomimetic search for material-structuring principles causing light, thermal and electrical interferences [55]. Their brilliant metallic appearance originates from structures in organic materials that are both electrically and thermally insulating. Thus, there is an industrial effort to develop insect cuticleinspired products with attractive metallic surfaces that do not feel so cold as their metallic counterparts and do not present an electrical shock hazard.
Figure 2
(a)
(b)
EV EP
Pr
ote
Pr
ot
ei n
in
HN
EXO N
N
Chitin/ Deacetylated chitin
HN
HO
?
Catechol
R
HO
NH
PCF N
MESO
NH
PC TcCPR27 Protein
ENDO
TcCPR18 TcCPR4 TcCP30
EC
Current Opinion in Insect Science
Proposed cross-linking network of cuticular proteins, chitin and catechols in rigid cuticle of a beetle. (a) Histidine residue(s) of CPs likely participate in cross-linking reactions through the quinone- and/or quinone methide intermediates derived from NADA and/or NBAD during cuticle maturation [7]. Addition adducts at either the 2-carbon and 6-carbon of the catechol ring or the b-carbon of the histidyl side chain with an imidazole nitrogen and/or the amino group of glucosamine are proposed to occur [56–58]. R COCH3 and COCH2CH2NH2 are NADA and NBAD, respectively. (b)Major CPs in rigid cuticle of T. castaneum. TcCPR27, TcCPR18 and TcCP30 are localized in both laminae and pore canal fibers, whereas TcCPR4 is predominantly localized in pore canal fibers [20,21,48]. TcCP30 undergoes cross-linking with TcCPR27 and TcCPR18, but not to TcCPR4 in rigid cuticle [48]. Abbreviations are listed in the legend of Figure 1. Current Opinion in Insect Science 2016, 17:1–9
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Cuticle formation and pigmentation in beetles Noh et al.
Current research is also focused on identifying the role(s) of individual CPs by following their expression profiles, localization and alterations in phenotypes as well as insect viability following depletion of specific cuticular components. Additional experiments are needed to determine the precise timing of appearance and location of individual components, their transport into and throughout the cuticle, and how they interact with one another to shape the overall architecture and properties of the exoskeleton. Why there is a need for such a large assortment of families of CPs and multiple members within each family and how they influence the properties of diverse cuticular structures will be the focus of future studies.
Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A2A01006614), and Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2015R1A6A3A04060323) to MYN.
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Current Opinion in Insect Science 2016, 17:1–9