Tyrosine Metabolism For Insect Cuticle Pigmentation and Sclerotization

Chapter 6
Tyrosine Metabolism for Insect Cuticle

Pigmentation and Sclerotization
Yasuyuki Arakane, Mi Young Noh, Tsunaki Asano, and Karl J. Kramer
Abstract Pigmentation or body color patterns in insects quite often differ not only
between species but also in different stages of development and in different body
regions of a single species. Body coloration plays physiologically and ecologically
important roles as for instance in species recognition and communication, court-
ship/mate selection, mimicry, crypsis, warning, prey-predator/parasite interactions,
and resistance to temperature, desiccation and absorbs or reflects harmful ultravio-
let radiation. Many kinds of pigment molecules and structural colors contribute to
the diversity of body coloration in insects. Recent studies have elucidated some of
the genetic and molecular biological mechanisms underlying pigment biosynthesis.
This chapter focuses on the pigments derived from the amino acid tyrosine. The
tyrosine-mediated cuticle tanning pathway is responsible for production of mela-
nins and other pigments derived from 3,4-dihydroxyphenylalanine (DOPA) and
dopamine as well as from N-acyldopamines. The N-acylated dopamines, in addi-
tion, are oxidized by the phenoloxidase laccase 2 to form quinones and quinone
methides, which then undergo cross-linking reactions with cuticular proteins (CPs)
for cuticle sclerotization. We review the regulation and functional importance and
also the diversity of the genes involved in this pathway. The unique localization and
cross-linking of specific CPs for morphology and ultrastructure of the exoskeleton
are also discussed.
Y. Arakane (*) • M.Y. Noh

Department of Applied Biology, Chonnam National University, Gwangju 61186, South Korea
e-mail: [email protected]; [email protected]
T. Asano
Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan
e-mail: [email protected]
K.J. Kramer
Department of Biochemistry and Molecular Biophysics, Kansas State University,
Manhattan, KS 66506, USA
e-mail: [email protected]
© Springer International Publishing Switzerland 2016 165

E. Cohen, B. Moussian (eds.), Extracellular Composite Matrices in Arthropods,
DOI 10.1007/978-3-319-40740-1_6
166 Y. Arakane et al.
6.1 Introduction
6.1.1 Insect Cuticle Composition and Morphology
The insect exoskeleton or cuticle is composed of multiple functional layers includ-

ing an outermost lipophilic waterproofing envelope, a protein-rich epicuticle and a
chitin/protein-rich procuticle that makes up the major portion (see Chap. 7 in this
book for details about insect hydrocarbons; Locke 2001; Moussian et al. 2006;
Moussian 2010). It plays critical roles in protecting insects from various physical
and environmental stresses and from pathogenic challenges. Two different struc-
tural biopolymers, cuticular proteins (CPs) and chitin (see Chap. 2 in this book for
details about chitin metabolism), are the major components of the exo- and endocu-
ticular layers that comprise the procuticle (Willis 2010). Other components include
pigments, catechols, mineral salts, lipids and water. The primary focus of this review
is the pigments found in insects that are derived from the amino acid tyrosine, which
include melanins and papiliochromes with the former being the predominant class
(Wittkopp and Beldade 2009; Shamim et al. 2014). Melanogenesis is a complex
multistep production of high molecular weight melanins via hydroxylation, oxida-
tion and polymerization of the oxidized metabolites (Singh et al. 2013). Although
melanin-type pigments commonly occur in insect cuticles, their isolation and partial
characterization have been carried out in only a few species (Hackman 1974, 1984;
Hori et al. 1984; Kayser 1985). No definitive molecular structure has yet been delin-
eated for these heterogeneous polymers, but they appear to be combinations of phe-
nolic, indolic, pyrrolic and aliphatic structures that may interact covalently and
noncovalently with macromolecular components such as cuticular proteins and chi-
tin (Schaefer et al. 1987; Duff et al. 1988; Solano 2014; Chatterjee et al. 2015).
There are several other kinds of pigments such as pterins, ommochromes, antra-
quinones, aphins (polycyclic quinones), tertapyrroles, carotenoids and flavonoids/
anthocyanins present in insect tissues, many of which have been described in earlier
reviews (Cromartie 1959; Fuzeau-Braesch 1972; Takeuchi et al. 2005; Shamim
et al. 2014). Ommochromes, for example, are one of the major pigments that have
been found in eyes, eggs and body walls of insects. In ommochrome biosynthesis,
tryptophan is converted to 3-hydroxykynurenine, which is then incorporated into
pigment granules by ABC transporters (Tearle et al. 1989; Pepling and Mount
1990). In addition, another transporter, which is a member of a major facilitator
superfamily, incorporates other precursor(s) into granules (Osanai-Futahashi et al.
2012b) where the pigments are synthesized via oxidative polymerization. Aphines
synthesized from a presumed polyketide precursor have only been found in aphids
and contribute to a variety of body colors during development and/or different spe-
cies. Tsuchida et al. (2010) demonstrated that infection with an aphid endosymbiont
of the genus Rickettsiella increases the amounts of blue-green aphins in the pea
aphid, Acyrthosiphon pisum, resulting in a change in their body color from reddish
to greenish. This body color change caused by endosymbiosis is likely to influence
prey-predator/parasite interactions and natural populations of the aphid. Pterins
6 Tyrosine Metabolism for Insect Cuticle Pigmentation and Sclerotization 167
synthesized from guanosine triphosphate are also widely distributed in eyes, bodies
and wings of insects. In addition, tetrahydrobiopterin (BH4) serves as a cofactor for
enzymes such as phenylalanine hydroxylase and tyrosine hydroxylase (TH) in tyro-
sine metabolism-associated cuticle pigmentation and sclerotization (Futahashi et al.
2010). For example, 6-pyruvoyl-tetrahydropterin synthase (PTS, Purple) is involved
in the BH4 biosynthesis, and a mutation in BmPTS gene is responsible for colorless
cuticle of the albino (al) mutant of the silkworm, Bombyx mori (Fujii et al. 2013).
Insects and mammals possess fine-tuned systems of enzymes to meet their
specific demands for tyrosine metabolites. In addition, more closely related enzymes
involved in tyrosine metabolism appear to have emerged in many insect species
(Vavricka et al. 2014). The metabolism of tyrosine plays a major role in not only the
darkening of insect cuticle but also in its hardening or sclerotization as well as in
innate immune responses to microbial pathogens. Coloration can vary from colorless
to yellow to tan to orange to brown to black depending on the amounts and types of
melanin-like pigments incorporated. The degree of sclerotization can vary from soft
and flexible to hard and stiff, much of which is determined by the number of chitin-
protein laminae and also the number of structural proteins’ cross-links derived from
tyrosine metabolism (Yang et al. 2014). The chemistry underlying insect
pigmentation is rather complex. However, substantial progress has been made in
recent years in understanding how tyrosine metabolism contributes to that process
(Sugumaran 2009; Shamim et al. 2014).
CPs and chitin are the major components of the exo- and endocuticular layers
that comprise the procuticle. The former layer is generally formed before molting,
whereas the latter is mainly deposited after completion of the molting process.
Transmission electron microscopic (TEM) analysis, for example, revealed that the
embryonic cuticle of the fruit fly, Drosophila melanogaster, as well as dorsal larval
body wall cuticle from the red flour beetle, Tribolium castaneum, are composed of
an envelope, epicuticle and procuticle, the latter consisting of various numbers of
horizontally oriented chitin-protein laminae parallel to the epidermal cell’s apical
plasma membrane (Fig. 6.1a; Moussian et al. 2006; Moussian 2010). These
morphologically distinct layers are also evident in cuticle of elytra (modified
forewings; Chen et al. 2015a, b) dissected from T. castaneum pharate adults, which
become harder and darker shortly after eclosion. In the case of elytral dorsal cuticle,
unlike the relatively soft and flexible elytral ventral and larval dorsal body wall
cuticles, there are numerous vertically oriented columnar structures denoted as pore
canal fibers (PCFs), which extend directly from the “apical plasma membrane
protrusions” (APMP) of the underlying epidermal cells and penetrate a large number
of horizontal laminae, reaching all the way to the epicuticle (Fig. 6.1b; Noh et al.
2014). Not only the horizontal laminae but also the vertical PCFs are likely
composed of chitin as those structures bind to wheat germ agglutinin (Noh et al.
2015b). In T. castaneum adults, other regions with rigid cuticle such as the thoracic
body wall and leg exhibit an ultrastructure very similar to that of the elytron’s dorsal
cuticle. On the other hand, there are fewer horizontal laminae and no vertical pore
canals with PCFs in anatomical regions covered with soft, flexible and less
pigmented cuticles such as those found on the dorsal abdomen, ventral elytron and
Fig. 6.1 Ultrastructure of larval (a) and adult body wall (b) cuticles in T. castaneum. Both larval
and adult body cuticles are composed of distinct layers including the envelope, epicuticle and
procuticle. The procuticle consists of a number of horizontal chitinous laminae in larval and adult
body wall cuticles. In addition, there are numerous pore canals running transverse to the laminae,
as well as to the apical plasma membrane. The canals extend from the apical plasma membrane to
the epicuticle region and contain a core of pore canal fibers in adult body cuticle. Ultrastructure of
adult body wall is similar to those of elytra and leg cuticles in T. castaneum, which are relatively
hard cuticles (Noh et al. 2014). EV envelope, EP epicuticle, PRO procuticle, PC pore canal, PCF
pore canal fiber, APMP apical plasma membrane protrusion. Scale bar = 2 μm
hindwing (Noh et al. 2014). Similar vertical fibrillar structures or vertical fibrils
have been observed not only in other insect species (Locke 1961; Delachambre
1971; Wigglesworth 1985) but also in the exoskeletons of crustaceans after removal
of minerals and some proteins, including Homarus americanus (American lobster),
Callinectes sapidus (Atlantic blue crab) and Tylos europaeus (sand-burrowing
isopod) (see Chap. 5 in this book for details about exoskeletons of crustaceans;
Raabe et al. 2006; Cheng et al. 2008; Seidl et al. 2011), suggesting that this unique
architecture and arrangement of numerous laminae and PCFs contribute to the
physical strength of rigid cuticle in arthropods. Pigments are found in cuticle and
epidermis and little or no information is available as to whether they contribute to
the mechanical properties of the exoskeleton.
6.1.2 Proposed Tyrosine-Mediated Cuticle Tanning Pathway
Despite a rather limited composition, cuticle has remarkably diverse mechanical

properties, ranging from soft and flexible to hard and rigid. An insect must periodi-
cally replace its old cuticle with a new one by undergoing ecdysis because the
mature cuticle is too restrictive to allow for continuous growth during development.
Immediately after molting, the cuticle is soft and pale, but it shortly becomes
hardened (sclerotized) and often darker (pigmented) over a period of several hours
or days. This vital process together with dehydration occurs during each stage of
development (Kramer et al. 2001; Andersen 2005; Arakane et al. 2008; Lomakin
et al. 2011).
Tanning (pigmentation and sclerotization) is a complex and important physio-
logical event not only in cuticle formation (Andersen 2010) but also in wound heal-
ing, encapsulation during a defensive response to infection of parasites, and
hardening of the egg chorion (see Chap. 9 in this book for details about chorion
hardening; Li 1994; Sugumaran 2002). With tyrosine as the initial substrate, the
cuticle tanning reactions include hydroxylation of tyrosine to dihydroxyphenylalanine
(DOPA) and decarboxylation of DOPA to dopamine. For melanization, furthermore,
oxidation of DOPA and dopamine to DOPA-quinone and dopaminequinone,
conversion of these quinones to dihydroxyindole (DHI) and/or 5,6-dihydroxyindole-
2-carboxylic acid (DHICA), oxidation of DHI and DHICA to DHI-chrome and
DHICA-chrome (melanochromes) and finally polymerization of melanochromes to
form melanins (Fig. 6.2; Arakane et al. 2009; Simon et al. 2009). For pigmentation
involving acylated quinones, the reactions include N-acetylation of dopamine to
N-acetyldopamine (NADA) or N-β-alanylation to N-β-alanyldopamine (NBAD),
oxidation of NADA and NBAD to NADA-quinone and NBAD-quinone and their
polymerization to form their corresponding pigments. These quinones, in addition,
undergo isomerization to quinone methides and cross-linking reactions with CP
side chains (most likely histidyl residues) for cuticle sclerotization (Fig. 6.2; Kramer
et al. 2001).
Recent studies have indicated that the following enzymes are involved in the
catalysis of cuticle tanning reactions: tyrosine hydroxylase (TH; Pale) converts
tyrosine to DOPA; DOPA decarboxylase (DDC) converts DOPA to dopamine;
dopachrome conversion enzyme (DCE, Yellow) accelerates the conversion of
dopachrome to DHI; arylalkylamine N-acetyltransferase (NAT) converts dopamine
to NADA; aspartate 1-decarboxylase (ADC) decarboxylates L-aspartic acid to
β-alanine for production of NBAD; NBAD synthase (Ebony) produces NBAD and
laccase 2 catalyzes the catecholic oxidation reactions in the tanning pathway (Table
6.1 and see Sect. 6.2 for details).
6.2 Functions of Key Enzymes/Proteins Involved in Tyrosine-

Mediated Cuticle Tanning
6.2.1 Tyrosine Hydroxylase (TH)
The first step in the cuticle tanning pathway is the hydroxylation of tyrosine to
produce DOPA (Fig. 6.2). There are two enzymes that can catalyze this hydroxylation
reaction, tyrosinase (phenoloxidase, PO) and tyrosine hydroxylase (TH). Although
the former enzyme has been detected in insect cuticles and is multifunctional,
Fig. 6.2 Proposed cuticle tanning pathway in T. castaneum. DOPA, 3,4-dihydroxyphenylalanine;

dopamine, 3,4-dihydroxyphenethylamine; NADA, N-acetyldopamine; NBAD, N-β-
alanyldopamine; TH, tyrosine hydroxylase; DDC, DOPA decarboxylase; NAT, N-acetyltransferase;
NBAD synthase (Ebony), N-β-alanyldopamine synthase; ADC, aspartate 1-decarboxylase;
NBADH (Tan), N-β-alanyldopamine hydrolase; Lac2, laccase 2; DCE (Yellow), dopachrome
conversion enzyme; CP, cuticle proteins. The broken and solid lines represent melanin synthesis
and quinone tanning pathways, respectively. Key enzymes are indicated in red letters and body
color changes are shown after RNAi for TcTH (a), TcDDC (b), TcLac2 (c), TcADC (d), Tcebony
(e), TcNAT (f), TcY-y (g) and TcY-e (h)
oxidizing ortho-diphenols as well as hydroxylating monophenols such as tyrosine

(Andersen 2005; Arakane et al. 2005; Kanost and Gorman 2008), it likely plays
only a role in immune-related melanization and not in cuticle tanning as suggested
by several studies (Barrett 1991; Marmaras et al. 1996; Ashida and Brey 1997;
Asano and Ashida 2001; Christensen et al. 2005; Kanost and Gorman 2008). In line
with this notion, double-stranded RNA (dsRNA)-mediated loss of function of
tyrosinase in T. castaneum, for instance, had no effect on larval, pupal and adult
cuticle tanning (Arakane et al. 2005).
Like mammalian TH, insect TH is a pterin (BH4)-dependent oxygenase (Liu
et al. 2010; Fujii et al. 2013) that catalyzes hydroxylation of tyrosine as a homotet-
ramer. Its activity is regulated by phosphorylation of a serine residue catalyzed by a
cAMP-dependent protein kinase (Vie et al. 1999). In D. melanogaster, there are two
alternatively spliced isoforms of DmTH (pale): the longer is expressed in the epider-
mis and the shorter, which lacks a highly acidic region in the N-terminus, specifi-
cally is expressed in the central nervous system (Birman et al. 1994; Friggi-Grelin
Table 6.1 Cuticle tanning-related genes and loss of function phenotypes in insects
Enzyme Species Accession no. Loss of function Phenotype References
Tyrosine hydroxylase Tribolium castaneum EF592178 RNAi Colorless and soft Gorman and Arakane
(TH) cuticle (2010)
Drosophila AAF50648 Mutant (pale) Unpigmented embryos/ Budnik and White
melanogaster pale cuticle (1987), Neckameyer
and White (1993),
and True et al. (1999)
Manduca sexta EF592177 – – Gorman et al. (2007)
Papilio xuthus AB178006 Chemical inhibition Suppression of larval Futahashi and
pigmentation Fujiwara (2005)
Pseudaletia separata AB274834 Chemical inhibition Diminished black Ninomiya et al.
pigmentation of larval (2006)
stripes
Bombyx mori AB439286 Mutant (sch)/RNAi Light reddish-brown Liu et al. (2010) and
neonate larval body Lee et al. (2015)
color/delay pupal
pigmentation
Spodoptera exigua JF795467 JF795468 Mutant (overexpression) Dark pigmented pupal Liu et al. (2015)
cuticle
6 Tyrosine Metabolism for Insect Cuticle Pigmentation and Sclerotization
Oncopeltus fasciatus KM247780 RNAi Loss of black Liu et al. (2014)

pigmentation in adult
cuticle
(continued)
171
172
Table 6.1 (continued)

Dopa decarboxylase Tribolium castaneum EU019710 RNAi Slightly darker adult Arakane et al. (2010)
(DDC) cuticle
Drosophila AAF53764 Mutant Albino adult bristle and True et al. (1999)
melanogaster cuticle
Pseudaletia separata AB072300 Chemical inhibition Diminished black Ninomiya et al.
pigmentation of larval (2006)
stripes
Papilio xuthus AB178007 Chemical inhibition Suppression of larval Futahashi and
pigmentation Fujiwara (2005)
Bombyx mori AF372836 RNAi Fail larval to pupal molt Wang et al. (2013)
Papilio glanucus AF036963 Mutant (melanic female) Yellow proximal area of Koch et al. (1998)
adult wing alters black
Spodoptera exigua SEU71404 Mutant (overexpression) Dark pigmented pupal Liu et al. (2015)
cuticle
Oncopeltus fasciatus KM247781 RNAi Reduction of adult black Liu et al. (2014)
pigmentation
Aspartate Tribolium castaneum EU019709 RNAi/ mutant (black and Black body Kramer et al. (1984)
1-decarboxylase (ADC) bchr/bST) pigmentation in larva, and Arakane et al.
pupa and adult (2010)
Drosophila NM_057440 mutant (black) Black/dark body Phillips et al. (1993,
melanogaster pigmentation 2005)
Bombyx mori KM523624 mutant (bp) Black pupal cuticle Dai et al. (2015)
Y. Arakane et al.
Arylalkylamine Tribolium castaneum FJ647798 RNAi Dark pigmented adult Tomoyasu et al.
N-acetyltransferase cuticle (2009)
(NAT) Bombyx mori DQ256382 RNAi/mutant (mln) Darker body Dai et al. (2010),
pigmentation Zhan et al. (2010),
and Qiao et al. (2012)
overexpression Dark coloration of Osanai-Futahashi
neonatal larvae, adult et al. (2012)
antenna and larval
tracheae alters light
brawn
NBAD synthase (Ebony) Tribolium castaneum FJ647797 RNAi Dark pigmented adult Tomoyasu et al.
cuticle (2009)
Drosophila AAF55870 mutant Dark pigmented adult Wittkopp et al.
melanogaster thorax and abdomen (2002a)
Papilio xuthus AB195255 – – Futahashi and
Fujiwara (2005)
Bombyx mori AB439000 Mutant (sooty) Dark body pigmentation Futahashi et al.
in larva and pupa (2008a, b)
Papilio glanucus – Mutant (melanic female) Black melanin replaces Koch et al. (2000)
yellow background in
wing
Papilio polytes AB525746 – – Nishikawa et al.
(2013)
(continued)
173
174

NBAD hydrolase (Tan) Drosophila NM_132315 Mutant Less pigmented adult True et al. (2005),
melanogaster thorax and abdomen Jeong et al. (2008),
and Aust et al. (2010)
Bombyx mori AB499125 Mutant (ro) Larval black marking Futahashi et al.
alters light brown (2010)
Papilio xuthus AB499122 – – Futahashi et al.
(2010)
Laccase 2 (Lac2) Tribolium castaneum AY884061 RNAi Colorless and soft Arakane et al. (2005)
cuticle, lethal
Monochamus EU093075 RNAi Colorless and soft Niu et al. (2008)
alternatus cuticle, lethal
Riptortus pedtris AB586067 RNAi Colorless and soft Futahashi et al.
cuticle, lethal (2011)
Nysius plebeius AB586069 RNAi Less pigmentation Futahashi et al.
(2011)
Megacopta AB586068 RNAi Less pigmentation Futahashi et al.
punctatissima (2011)
Papilio xuthus AB499123 – – Futahashi et al.
(2010)
Oncopeltus fasciatus KM247782 RNAi Reduction of adult black Liu et al. (2014)
pigmentation
Nilaparvata lugens KR086787 RNAi Colorless and soft Ye et al. (2015)
cuticle, lethal
Y. Arakane et al.
Yellow-y (Y-y) Tribolium castaneum GU111770 RNAi Not critical for body Arakane et al. (2010)
coloration except for
black pigmented
pterostigma of the
hindwing
Drosophila NM_057444 mutant Light brown/yellowish Wittkopp et al.
melanogaster cuticle (2002a)
overexpression Black adult wing cuticle Riedel et al. (2011)
Bombyx mori AB438999 mutant (ch) Lighter pigmented larval Futahashi et al.
cuticle (2008a, b)
Papilio xuthus AB195254 – – Futahashi and
Fujiwara (2007)
Yellow-e (Y-e) Tribolium castaneum GU111765 RNAi Dark body color in adult Noh et al. (2015a)
body cuticle and vein of
hindwing
Bombyx mori AB489224 mutant (bts) White larval head and Ito et al. (2010)
anal plates alters reddish
brown
175
et al. 2003). Similarly, long epidermal and short brain/neural isoforms of the TH
transcript were identified in the oriental armyworm, Pseudaletia separata (Ninomiya
and Hayakawa 2007).
Several studies have implicated a functional importance of TH in insect cuticle
pigmentation. Mutation in the DmTH gene (pale) results in lethal unpigmented
embryos that fail to develop into larvae, resulting in no hatching from eggs (Budnik
and White 1987; Neckameyer and White 1993). Because TH is also important in the
nervous system, it is possible that the embryos die before becoming fully developed
because of the lack of DmTH function in this system. This embryonic lethality was
rescued by expression of the epidermal form of DmTH (Friggi-Grelin et al. 2003).
True et al. (1999) demonstrated by using mosaic analysis that the lack of DmTH
function resulted in an adult albino cuticle phenotype, and ectopic DmTH expression
caused ectopic cuticle pigmentation. In lepidopteran species such as the Asian
swallowtail, Papilio xuthus and P. separata, TH mRNA and/or protein were detected
in the epidermal cells underlying the darkly marked larval cuticle (Futahashi and
Fujiwara 2005; Ninomiya and Hayakawa 2007). In the tobacco hornworm, Manduca
sexta, the amount of TH protein in the integument of pharate pupal segments is
correlated with the degree of cuticle pigmentation (Gorman et al. 2007). Treatment
with a TH inhibitor such as 3-iodotyrosine inhibited pigment formation in P. xuthus
larvae and B. mori adults (Futahashi and Fujiwara 2005; Lee et al. 2015) and also
rescued black pupae produced by overexpressed TH and DDC genes in a pupal
melanic mutation strain of the beet armyworm, Spodoptera exigua (Liu et al. 2015).
In addition, RNAi for BmTH caused a delay in pupal cuticle pigmentation of B. mori
(Lee et al. 2015), and a reduction of BmTH transcripts was responsible for the sex-
linked chocolate (sch) mutant, which exhibits a light reddish-brown neonatal larval
body color compared with the black color of that of the wild-type strain (Liu et al.
2010). There is differential regulation of TH during cuticular melanization and
innate immunity in B. mori (Lee et al. 2015). BmTH is expressed in the epidermis
during development for the purpose of pupal cuticle melanization and pigmentation
in adults, and in the fat body during infection for antimicrobial activity. In the large
milkweed bug, Oncopeltus fasciatus, RNAi for the OfTH gene resulted in a complete
absence of black pigmentation in adult cuticles on the head, thorax, abdomen and
wings (Liu et al. 2014). Similarly, in T. castaneum, injection of dsRNA for TcTH
diminished the brown pigmentation in the pupal cuticle of the abdominal segments,
urogomphi, bristles and gin traps as well as in the adult cuticle of the mandibles and
legs, which was visible underneath the pupal cuticle, and also the dark pigmentation
observed in the hindwings. The cuticle of phypomorphic dsTcTH-treated adults is
pale (Fig. 6.2a; Gorman and Arakane 2010). Furthermore, the cuticles of both hard
and dark dorsal thorax and hard and colorless eye of dsTcTH-treated adults are soft
and flexible, indicating that TH is required for not only cuticle pigmentation but also
for sclerotization in some species.
6.2.2 Dopa Decarboxylase (DDC)
Dopamine is a catecholamine involved in nervous systems of mammals and insects

as a neurotransmitter, neuromodulator and neurohormone (Nassel 1996; Osborne
1996; Neckameyer and Leal 2002; Han et al. 2010). In insects, dopamine is also
important for egg-shell hardening and the immune response as well as cuticle
melanization (Hopkins et al. 1984; Nappi et al. 1992; Huang et al. 2005; Davis et al.
2008; Paskewitz and Andreev 2008; Sideri et al. 2008). In the cuticle tanning
pathway, DOPA decarboxylase (DDC), which is a pyridoxal-5-phosphate (PLP)-
dependent enzyme, catalyzes the decarboxylation of DOPA to yield dopamine, a
major precursor in melanin/pigment production and sclerotization mediated by
protein cross-linking (Hopkins et al. 1984; Hiruma et al. 1985; Kramer and Hopkins
1987; Andersen 2005; Hopkins and Kramer 1992; Riddiford et al. 2003).
The functional importance of DDC in cuticle coloration has been well studied in
several lepidopteran species, which exhibit a high degree of variation in pigmentation.
Like the TH gene, DDC is highly expressed in epidermal cells underlying the
presumptive black markings (e.g. eyespots, V-shaped and band-markings) of larval
cuticles of different swallowtail butterflies including P. xuthus, the common yellow
swallowtail, Papilio machaon and the common Mormon, Papilio polytes (Shirataki
et al. 2010). Furthermore, chemical inhibitors of DDC such as
m-hydroxybenzylhydrazine (HBHZ) completely inhibited pigment formation of P.
xuthus (Futahashi and Fujiwara 2005). In the eastern tiger swallowtail, Papilio
glanucus, DDC expression and enzyme activity regulate the color pattern of the
adult wings (Koch et al. 1998). DDC mRNA and activity are detected early in the
presumptive yellow regions of the wings and later in the presumptive black patterns.
In the melanic female of this species, early DDC activity in the central yellow region
of the wing is much lower than that of wild-type females, which is later melanized
concomitant with increased DDC activity. Ninomiya et al. (2006) demonstrated that
DDC expression is required for the black strips in the dorsal cuticle of last instar
larvae of P. separata. DDC mRNA and protein are detected in the epidermal cells
underneath the black stripe, but not below the white stripe. HBHZ treatment caused
the complete loss of DDC activity, a low level of dopamine, an abnormally high
level of DOPA and diminished black pigmentation of the strips in the larval cuticle,
indicating that DDC activity and its product dopamine are critical for melanin
deposition in the black strips.
In D. melanogaster, the DmDDC gene has two alternatively spliced transcripts,
of which one is expressed in the epidermis and the other in the central nervous
system (Hodgetts and O’Keefe 2006). Tissue-specific expression of alternatively
spliced DDC isoforms has not been reported in other species. The crystal structure
and site-directed mutagenesis analysis of DmDDC indicate that T82 is involved in
substrate binding and H192 is essential for both a substrate interaction and cofactor
binding (Han et al. 2010). These amino acid residues are highly conserved among
insect DDCs. Like that observed for DmTH, patches of epithelial cells deficient in
DmDDC activity produce albino adult bristles and cuticle (True et al. 1999).
Similarly, loss of function of DDC by RNAi causes a reduction in black pigmentation

of O. fasciatus adult cuticle (Liu et al. 2014). In T. castaneum, the level of DOPA
increases approximately 5-fold in dsTcDDC-treated pharate adults (Arakane et al.
2009). However, the initial cuticle pigmentation of the resulting adults is substantially
delayed, suggesting that, unlike dopamine, DOPA does not appear to be utilized to
a great extent for DOPA quinone-associated melanin synthesis, probably because
DOPA is a poor substrate for laccase 2 (Arakane et al. 2009). The body color of the
TcDDC-deficient mature adults is slightly darker than that of control animals (Fig.
6.2b). This phenotype may be due to a small amount of DOPA-melanin accumulation
relative to NBAD- and/or NADA-derived pigments in the adult cuticle. All of these
results indicate that DDC activity is required for providing dopamine as a major
precursor for melanin synthesis and also quinone-based tanning in insect cuticle.
6.2.3 Aspartate 1-Decarboxylase (ADC)
β-Alanine is involved in critical physiological events in insects. In cuticle tanning,

it is conjugated with dopamine via the action of NBAD synthase (ebony; see Sect.
6.2.5) to produce NBAD, which is one of the major catechols serving as a cuticle
tanning precursor (Hopkins et al. 1984; Kramer et al. 1984; Andersen 2007, 2010).
β-Alanine is produced by decarboxylation of the α-COOH of aspartic acid, and
aspartate 1-decarboxylase (ADC) catalyzes this reaction. Like DDC, ADC is a
member of the pyridoxal 5-phosphate-dependent amino acid decarboxylase family,
in which all members possess a conserved decarboxylase domain containing a
pyridoxal phosphate-binding domain motif (Ser-X-X-Lys). Phylogenetic analysis
revealed that ADCs are closely related with glutamate decarboxylases (GDCs) and
distantly related with other decarboxylase family members including histidine,
tyrosine and DOPA decarboxylases (Fig. 6.3; Arakane et al. 2009). Although ADCs
and GDCs show a high overall amino acid sequence identity/similarity, they exhibit
a rather rigorous substrate specificity probably due to small differences in the
enzymes’ active sites. Richardson et al. (2010) demonstrated that the recombinant
ADC protein from the yellow fever mosquito, Aedes aegypti, produces β-alanine by
decarboxylation of aspartic acid, but it is inactive toward glutamic acid. On the
other hand, the recombinant GDC from the malaria mosquito, Anopheles gambiae,
decarboxylates glutamic acid to produce γ-amino butyric acid but exhibits no
activity toward aspartic acid. In addition, that research group identified Q377 to be
located in the active site of Ae. aegypti ADC, which is a highly conserved amino
acid residue among other insect ADCs and appears to be critical for selectivity of
aspartic acid as the substrate (Liu et al. 2012) (Table 6.2).
In D. melanogaster, the black gene encoding ADC (DGAD2) is responsible for
black/dark body color phenotype (Phillips et al. 1993, 2005). The black mutant had
a deficiency of β-alanine (Hodgetts and Choi 1974) probably due to a significant
decrease in ADC activity (Phillips et al. 2005). Similarly, black body color mutant
100 PmDDC
86 PxDDC
100 BmDDC
SeDDC
65 99 MbDDC
92 PsDDC Dopa
TcDDC decarboxylase
100
100 TmDDC
DmDDC
AaDDC
98
43
93 AsDDC
99 AgDDC
BmHDC
TcHDC
100 CqHDC
87
100 99 AgHDC
93 Histidine
DmHDC decarboxylase
82 NvHDC
100 AmHDC
100 BiHDC
DmTDC Tyrosine
99 TcTDC decarboxylase
56 ObADC
54 BbADC
63 BmADC
100
BaADC Aspartate
45
DpADC 1-decarboxylase
68
TcADC
100
AaADC
DmADC
100 BmGDC
PxGDC
100 AmGDC
100 BtGDC
TcGDC
92 Glutamate
45 DmGDC
97
decarboxylase
67 BdGDC
MdGDC
97 AgGDC
99 AaGDC
78 CqGDC
Fig. 6.3 Phylogenetic analysis of amino acid decarboxylases in insects. The amino acid sequences
of DOPA decarboxylases (DDC), histidine decarboxylases (HDC), tyrosine decarboxylases
(TDC), aspartate 1-decarboxylases (ADC) and glutamate decarboxylases (GDC) were obtained
from GenBank. The phylogenetic tree was constructed with MEGA 6.06 software using the
Neighbor-Joining method (Tamura et al. 2013). Numbers by each branch indicate results of
bootstrap analysis of 5000 replications. See Table 6.2 for the accession numbers of protein
sequences used in this analysis
Table 6.2 Accession numbers of insect decarboxylases used for phylogenetic analysis
Decarboxylase Abbreviation Species Accession number
Dopa decarboxylase PmDDC Papilio machaon BAJ07588
(DDC) PxDDC Papilio xuthus NP_001299156
BmDDC Bombyx mori NP_001037174
SeDDC Spdoptera exigua AFG25780
MbDDC Mamestra brassicae BAB68545
PsDDC Pseudaletia separata BAB68549
TcDDC Tribolium castaneum ABU25222
TmDDC Tenebrio molitor BAA95568
DmDDC Drosophila melanogaster NP_724164
AaDDC Aedes aegypti AAC31639
AsDDC Anopheles sinensis KFB39134
AgDDC Anopheles gambiae XP_319841
Histidine decarboxylase BmHDC Bombyx mori XP_012551888
(HDC) TcHDC Tribolium castaneum XP_975682
CqHDC Culex quinquefasciatus EDS34715
AgHDC Anopheles gambiae EAA14857
DmHDC Drosophila melanogaster CAA49989
NvHDC Nasonia vitripennis XP_008202389
AmHDC Apis melifera XP_392129
BiHDC Bombus impatiens XP_012245143
Tyrosine decarboxylase DmTDC Drosophila melanogaster AAM70810
(TDC) TcTDC Tribolium castaneum EFA10808
Aspartate 1-decarboxylase ObADC Operophtera brumata KOB74239
(ADC) BbADC Biston betularia AEP43793
BaADC Bicyclus anynana AEQ77286
DpADC Danaus plexippus EHJ66406
TcADC Tribolium castaneum ABU25221
AaADC Aedes aegypti EAT40747
DmADC Drosophila melanogaster NP_476788
BmADC Bombyx mori AJQ30182
Glutamate decarboxylase BmGDC Bombyx mori XP_004925034
(GDC) PxGDC Papilio xuthus KPI96691
AmGDC Apis melifera XP_391979
BtGDC Bombus terrestris XP_012170748
TcGDC Tribolium castaneum EFA06442
DmGDC Drosophila melanogaster CAA53791
BdGDC Bactrocera dorsalis XP_011201397
MdGDC Musca domestica XP_011296520
AgGDC Anopheles gambiae EAA11955
AaGDC Aedes aegypti EAT35903
CqGDC Culex quinquefasciatus EDS27073
strains of T. castaneum exhibit decreased concentrations of both β-alanine and

NBAD as well as a higher level of dopamine when compared with those of wild-
type beetles (Kramer et al. 1984). RNAi for TcADC resulted in a dark pigmented
body color (Fig. 6.2d) and a significantly lower NBAD content compared to control
animals like that seen in the T. castaneum black mutant strains (Arakane et al. 2009).
Injection of β-alanine, the expected product of the reaction catalyzed by ADC, into
the black mutant strains and dsTcADC-treated animals rescued the black body color
phenotypes and restored the normal reddish-brown cuticle coloration (Kramer et al.
1984; Arakane et al. 2009). In addition, the black mutation affected the puncture
resistance of the cuticle by delaying sclerotization (Roseland et al. 1987).
Furthermore, dynamic mechanical analysis indicated less cross-linked cuticles from
the black body color mutant and dsTcADC-treated animals (Arakane et al. 2009).
These results suggested that the black mutants of T. castaneum, like the D.
melanogaster black mutant, have a mutation(s) that causes loss of function of ADC,
resulting in a depletion of β-alanine used for NBAD synthesis. Solid-state 13C-nuclear
magnetic resonance difference spectroscopy was used to determine the presence of
melanin in the black mutant of T. castaneum (Kramer et al. 1989, 1995). It was
estimated that 1–2 % of the organic components in the cuticle of the black mutant
are attributable to eumelanin. The high levels of dopamine relative to the
corresponding levels in the wild-type strain led to an increased production of
eumelanin when the excess dopamine was oxidized in the black mutant (Kramer
et al. 1984; Roseland et al. 1987).
Recently, Dai et al. (2015) reported that a mutation of the ADC gene (BmADC)
is responsible for the black pupal (bp) mutant phenotype of B. mori, which exhibits
melanization specifically in the pupal stage. In the bp mutant, like that seen in the
black mutants of T. castaneum, there were depleted levels of BmADC transcripts,
β-alanine and NBAD as well as accumulation of dopamine. Injection of β-alanine
into the bp mutant reverted the dark color pattern to the wild-type pattern. RNAi for
BmADC in the wild-type strain, furthermore, led to a melanic pupal phenotype
similar to the bp mutant. All of these results indicated that ADC plays a role in
cuticle pigmentation and sclerotization. Loss of function of ADC results in a
depletion of β-alanine used for NBAD synthesis resulting in the accumulation of
abnormally high levels of dopamine, which are then used for dopamine melanin
production during tanning at the expense of NBAD quinone-mediated cuticle
protein cross-linking and pigmentation.
6.2.4 Arylalkylamine N-Acetyltransferase (AANAT)
Arylalkylamine N-acetyltransferases (AANATs) belong to a large Gcn5-related

acetyltransferase (GNAT) superfamily, which catalyze the transacylation between
acetyl-CoA and arylalkylamines (Dyda et al. 2000; Vetting et al. 2005). AANAT has
been extensively studied as a key enzyme for pineal hormone melatonin synthesis,
which regulates circadian rhythms in mammals (Arendt et al. 1995; Evans 1989;
Klein 2007). AANAT activity is rate-limiting and it acetylates serotonin to form

N-acetylserotonin in the vertebrate pineal organ, which is then methylated by
hydroxyindole-o-methyltransferase to melatonin.
In contrast to mammals, the availability of whole genome sequences from several
insect species has revealed that there is a large number of genes encoding AANAT
or AANAT-like proteins in their genomes (Mehere et al. 2011; Han et al. 2012;
Barbera et al. 2013; Hiragaki et al. 2015), suggesting a functional diversity of
AANATs in insects. For example, melatonin has also been detected in insects such
as D. melanogaster, B. mori, A. pisum, the migratory locust, Locusta migratoria,
and the American cockroach, Periplaneta americana (Vivien-Roels et al. 1984;
Finocchiaro et al. 1988; Hintermann et al. 1995; Itoh et al. 1995a, b; Hardie and Gao
1997). AANAT activity in melatonin synthesis was correlated with a circadian
rhythm and seasonal photoperiodism (Itoh et al. 1995b; Bembenek et al. 2005;
Vieira et al. 2005; Barbera et al. 2013). In mammals, several neurotransmitter
arylalkylamines (e.g. octopamine, dopamine and serotonin) are inactivated by
monoamine oxidase (MAO), whereas AANATs appear to metabolize arylalkylamines
in insects because there is little or no MAO activity in their nervous tissues (Smith
1990; Amherd et al. 2000; Sloley 2004).
In insect cuticle tanning, AANAT apparently N-acetylates dopamine to form
NADA, which is one of major precursors for quinone-mediated pigmentation and
sclerotization (Fig. 6.2; Andersen 1974, 2005, 2010; Hopkins and Kramer 1992).
Although the properties of insect AANATs including pH-activity profile, substrate
specificity, kinetic parameters and site-direct mutagenesis to identify residues that
participate in its catalysis have been studied (Hintermann et al. 1996; Brodbeck
et al. 1998; Mehere et al. 2011; Han et al. 2012; Dempsey et al. 2014), information
about the functional importance of AANATs in cuticle tanning is rather limited. In
B. mori, the melanism (mln) mutant, in which the AANAT (Bm-iAANAT) gene is
disrupted, shows a darker body pigmentation in the head, thoracic legs, spiracle,
claw hook of prolegs and anal plate of larvae as well as in the entire body of adults
(Dai et al. 2010; Zhan et al. 2010). Confirmation that a mutation of the Bm-iAANAT
was responsible for a dark color integument in the mln mutant was obtained by an
RNAi experiment. Injection of dsRNA for Bm-iAANAT into wild-type pupae resulted
in the adults exhibiting a darker body pigmentation similar to that of the mln mutant
(Zhan et al. 2010). In contrast, ectopic expression of Bm-iAANAT altered the dark/
black coloration of neonatal larvae and adult antennae as well as larval tracheae of
B. mori to light brown (Osanai-Futahashi et al. 2012a). This coloration change was
also evident in the B. mori black striped strain striped, which has a wide black stripe
in each segment. The black stripes became light grey in Bm-iAANAT-overexpressing
lines. All of these results support the hypothesis that N-acetylation of dopamine
decreases the availability of dopamine for dopamine-melanin production.
The dopamine content in the dark pigmented tissues (e.g. head, thoracic legs and
anal plate) from the mln mutant of B. mori was two times higher than that from the
wild-type strain (Dai et al. 2010). More recently, the same research group reported
additional information about the catecholamine content in the mln mutant (Qiao
et al. 2012). In the head of the larvae, dopamine and NBAD levels were higher than
those of the wild-type. In the whole body of adults, dopamine content was approxi-
mately six times greater than in the wild-type, while NBAD content was nearly the
same in the two strains. This may be due to significantly lower levels of ADC and
NBAD synthase (ebony) present in the adult mln mutant. Little or no NADA was
evident in both larval and adult tissues. These results suggest that a loss of function
of Bm-iAANAT results in excess dopamine (and NBAD in larvae), which likely
undergoes dopamine-melanin synthesis (and NBAD-pigment synthesis in larvae) in
the B. mori mln mutant. Dynamic mechanical analysis revealed that elytra with
abnormally high dopamine collected from the TcADC-deficient adult T. castaneum
(dsTcADC knockdown and the black mutants) exhibited a higher elastic modulus,
suggesting a less effectively cross-linked cuticle than that of control insects (Arakane
et al. 2009; Lomakin et al. 2010, 2011). In contrast, the wings of the mln mutant
showed a higher modulus, indicating that the mln wings are stiffer than those of the
wild-type strain, probably because the CPs are abnormally cross-linked.
Similarly, in adult T. castaneum, depletion of TcAANAT1 function by RNAi
resulted in dark pigmentation of the entire body including pronotum, ventral
abdomen, elytron and veins of the hindwing (Tomoyasu et al. 2009). In addition,
dark pigments surrounding the bristles located on the inter-veins of the elytron were
evident (Fig. 6.2f). The elytron is a highly modified and sclerotized forewing of
beetles. There are a large number of pillar-like support structures called “trabeculae”,
which are located between the dorsal and ventral cuticles and contribute to the
mechanical strength of the elytron (Ni et al. 2001; Chen and Wu 2013; Chen et al.
2015b; He et al. 2015; van de Kamp et al. 2015). The dark pigments in the elytra of
dsTcAANAT-treated adults appear to be due to those pigmented trabeculae
(unpublished observation), suggesting that NADA-mediated pigmentation and/or
sclerotization is required for development of the trabeculae.
6.2.5 NBAD Synthase (Ebony)
The gene for Ebony encodes an enzyme catalyzing the synthesis of NBAD by conju-
gation of dopamine and β-alanine. The ebony mutant of D. melanogaster had already
been described for its characteristic dark cuticle as early as 1923 (Bridges and
Morgan 1923). Mutants in the ebony locus also show phenotypes of altered locomo-
tion rhythm, vision or courtship behavior (Hotta and Benzer 1969; Kyriacou et al.
1978; Newby and Jackson 1991; Suh and Jackson 2007). Ebony (NBAD synthase) is
closely related to non-ribosomal peptide synthases (NRPSs). Study of recombinant
Ebony proteins of D. melanogaster that were expressed in an E. coli system revealed
its biochemical properties (Richardt et al. 2003). D. melanogaster Ebony consists of
879 amino acids, which is divided into three domains, an activation/adenylation
domain (572 aa), thiolation domain (78 aa) and amine-selecting domain (229 aa)
(Hovemann et al. 1998; Richardt et al. 2003; Hartwig et al. 2014). The selectivity of
amines is not very strict, and various biogenic amines, including dopamine, octopa-
mine, histamine and serotonin, are β-alanylated (Richardt et al. 2003).
NBAD synthase can be defined with a more general name, β-alanylbiogenic

amine synthase. Ebony is a novel type of NRPS-related protein that can be
distinguished by its ability to rapidly conjugate the activated β-alanine and biogenic
amines. The C-terminal amine selecting domain in Ebony is not homologous to any
other protein with known domains, indicating a specific structural selection of the
biogenic amine substrates (Hartwig et al. 2014). ebony is expressed in epithelial
cells during cuticle sclerotization. In the visual system of D. melanogaster, ebony
expression is localized in neural tissues, exclusively the neuropile and epithelial
glial cells (Richardt et al. 2002, 2003). Its expression can be detected also in many
regions of the brain and ventral nervous system from both larval and adult stages
(Suh and Jackson 2007). In photoreception, it is thought that Ebony is regulating the
neurotransmitter through the inactivation of histamine by N-β-alanylation (Richardt
et al. 2002, 2003).
The involvement of Ebony in cuticle hardening is supported by a tradeoff
relationship between the amount of NBAD and cuticle hardness, as showed in an
analysis of the German cockroach, Blattella germanica (Czapla et al. 1990). This
relationship is also demonstrated in the black mutant or ADC knockdown of T.
castaneum such that the decreased amount of NBAD correlates with lowered levels
of cross-links in the elytral cuticle (see Sect. 6.2.3; Arakane et al. 2009). A
biochemical characterization of the ebony mutant was performed by demonstrating
the incorporation of isotopically labeled β-alanine or dopamine into the pupal case
or by examining the use of sources for NBAD synthesis, including β-alanine,
dopamine and uracil, during cuticle formation (Jacobs 1968; Hodgetts 1972;
Hodgetts and Konopka 1973; Hodgetts and Choi 1974). In both the ebony mutant of
D. melanogaster and a melanotic body color mutant of the medfly, Ceratitis capitata,
it was postulated that a defective NBAD synthase is responsible for the low level of
NBAD and conversely high level of dopamine leading to a darker coloration (Wright
1987; Wappner et al. 1996). By using an in vitro cell free system of tissue homogenate
from C. capitata, the enzyme activities of Ebony were directly characterized.
Endogenous substrates like dopamine, norepinephrine and L-tyrosine were
β-alanylated by tissue extracts from wild-type individuals, but the extract from the
mutant exhibited only negligible levels of β-alanylation of these substrates (Perez
et al. 2002). No N-β-alanyldopa was synthesized, which is consistent with the
absence of N-β-alanyldopa in insect cuticle. It was reported in studies of Sarcophaga
species that N-β-alanyl-tyrosine (Sarcophagine) and a derivative of N-β-alanyldopa,
N-β-alanyl-5-S-glutathionyl-3-4-dihydroxyphenylalanine (5-S-GAD), have
antibacterial activities (Leem et al. 1996; Meylaers et al. 2003). NBAD itself has an
antibacterial activity comparable in strength to those of N-β-alanyldopa and 5-S-
GAD (Schachter et al. 2007). The NBAD levels were increased in the hemolymph
of the mealworm beetle, Tenebrio molitor, after bacterial challenges (Kim et al.
2000). Furthermore, NBAD synthase activity was detected in the integument of
bacteria-injected T. molitor larvae (Schachter et al. 2007). In C. capitata, expression
of ebony was induced in epidermal cells in response to bacterial challenges. After a
bacterial challenge, an allele of the ebony mutant (e11) with a cuticle deficiency
(Lindsley and Grell 1968) showed susceptibility to Serratia infection via oral
administration (Flyg and Boman 1988). These observations imply a direct involve-
ment of NBAD in defense reactions or probably in protection by a mechanically
strong cuticle that is stabilized by protein cross-links involving NBAD.
Like other genes involved in pigmentation, insect body color pattern is associ-
ated with the pattern of ebony expression. NBAD is also important for production
of papiliochromes that are yellowish-reddish-brownish pigments composed of
NBAD and kynurenines in Papilionidae butterflies (Umebachi 1990). In larvae of P.
xuthus, ebony is expressed in the epithelial cells underneath those cuticle regions
that have a reddish-brown coloration (Futahashi and Fujiwara 2005). Expression of
ebony is observed in red and yellow regions of the wings of P. polytes (Nishikawa
et al. 2013). In Drosophilid species, high levels of ebony expression are linked to
low levels of pigmentation in the thoracic trident or abdomen (Wittkopp et al. 2002a,
2009; Pool and Aquadro 2007; Takahashi et al. 2007). The same correlation was
observed with the colors of butterfly wings (Ferguson et al. 2011b). In B. mori sooty
mutants with a dark body color, ebony was identified as the responsible gene
(Futahashi et al. 2008b). Recently, Ebony was utilized as a visible marker gene for
genotyping of transgenic insects. Ubiquitous expression of ebony via the Gal4/UAS
system in B. mori caused a light pigmentation in the larval body or adult antennae
(Osanai-Futahashi et al. 2012a).
6.2.6 NBAD Hydrolase (Tan)
NBAD hydrolase (Tan) is a product of the tan gene and it shows high sequence
similarity to fungal isopenicillin N-acyltransferase (IAT; True et al. 2005), which is
involved in penicillin-G biosynthesis (Queener and Neuss 1982; Barredo et al.
1989). Like IAT, Tan appears to be expressed as a precursor protein that is activated
by self-processing into two polypeptide subunits at a conserved Gly-Cys motif (e.g.
Gly121-Cys122 in DmTan; Wagner et al. 2007; Aust et al. 2010; Perez et al. 2011).
One of the essential roles of Tan activity is the hydrolysis of β-alanylhistamine
(carcinine) in the visual system (True et al. 2005; Wagner et al. 2007; Perez et al.
2011). In D. melanogaster, DmTH is localized in photoreceptor cells where it
hydrolyzes carcinine to histamine for neurotransmission with the former metabolite
provided by NBAD synthase (Ebony) localized in the surrounding glial cells
(Richardt et al. 2002).
In insect epidermal cells, Tan hydrolyzes NBAD to form dopamine and β-alanine,
which is a reverse reaction of NABD synthesis catalyzed by Ebony (Fig. 6.2;
Wittkopp et al. 2002a; Wright 1987; True et al. 2005). A functional importance of
tan in cuticle pigmentation has been reported for a few insect species including D.
melanogaster, B. mori and P. xuthus. Disruption of the tan gene by mutation or
P-element insertion caused pigmentation defects in thoracic and abdominal cuticles
in adult D. melanogaster (True et al. 2005; Jeong et al. 2008), and ectopic expression
of tan rescued the pigmentation phenotypes in the tan mutant (True et al. 2005). In
addition, Jeong et al. (2008) reported that expression of both tan and dopachrome
conversion enzyme (yellow, see Sect. 6.2.8) genes correlated with a diversity of
body pigmentation patterns between D. yakuba and D. santomea. In the latter, the
loss of abdominal pigmentation involves little or no tan and yellow gene expression.
In P. xuthus and B. mori, expression of tan along with laccase 2 (see Sect. 6.2.7)
is strongly correlated with larval black markings/pigments (Futahashi et al. 2010).
For example, the tan transcript was clearly detected in the black region of the
eyespot marking of P. xuthus larvae, but it was absent in the reddish-brown region
of this marking. In the B. mori rouge (ro) mutant, the larval black markings are light
brown. In this mutant, the tan cDNA lacks exon 2, resulting in a premature stop
codon insertion. The predicted Tan protein is missing a large portion including a
conserved self-processing site, suggesting that tan is responsible for the larval body
color phenotype in the ro strain (Futahashi et al. 2010). Taken together, these results
suggest that Tan plays a role in cuticle pigmentation through hydrolysis of NBAD
to provide dopamine, which is a major precursor for melanin production. Tan may
also be critical for the cuticle’s mechanical properties because its activity influences
the level of NBAD, which serves as a cuticle protein cross-linking agent. Further
study is required to confirm this hypothesis.
6.2.7 Laccase (Lac)
Since the first description of laccase from the Japanese lacquer tree, Rhus vemic-
ifera (Yoshida 1883), this enzyme has been extensively studied, and now there is a
large accumulation of knowledge about its enzymatic properties, gene function and
structure (Nakamura and Go 2005; Sharma et al. 2007). Proteins like laccase, ascor-
bate oxidase or the bacterial proteins, CueO or CumA, have a common feature in
that these proteins are composed of three repeats of cupredoxin domains. They form
a sub-protein group of three domain multicopper oxidases (3dMCO). In insects,
laccase is regarded as one of the key enzymes for cuticle sclerotization and pigmen-
tation. The roles of laccase are thought to be dependent on its enzyme activity to
oxidize ortho-diphenols to the corresponding quinones. Since the first characteriza-
tion of laccase-like activity in Drosophila virilis (Ohnishi 1954; Yamazaki 1969),
laccase-like proteins were partially purified from the integument from several insect
species (Yamazaki 1972; Andersen 1978; Barrett and Andersen 1981; Barrett 1987a,
b; Thomas et al. 1989). Until the end of 1980s, the enzymatic properties of these
proteins were characterized in some detail (Ashida and Yamazaki 1990; Barrett
1991; Dittmer and Kanost 2010). In 2004, the first cDNA sequences for laccase-like
proteins from lepidopteran and dipteran species were reported (Dittmer et al. 2004).
In this study, three genes, laccase 1 and laccase 2 from M. sexta (MsLac1 and
MsLac2) and laccase 1 (AgLac1) of An. gambiae were identified. One characteristic
of the insect laccase-like proteins is a methionine residue at the T1 copper center
(Met716 of MsLac1, Met728 of MsLac2 and Met948 of AgLac1), whereas in lac-
cases from plants and fungi this residue position has a phenylalanine or leucine.
Other characteristics of the insect laccase-like proteins are the N-terminal exten-
sions that include an N-terminal signal peptide and a cysteine-rich region in all three
of the proteins. By using the efficient RNAi system of T. castaneum, loss of function
phenotypes were analyzed. dsRNA for laccase 2 (TcLac2) was injected into the
hemocoel of individuals at various stages. After ecdysis to larva, pupa and adult, the
new cuticles of the dsRNA-injected individuals were untanned. Also in the adult, a
severe wing deformation was also observed (Arakane et al. 2005).
By the early 1990s, three types of phenol-oxidizing enzymes had been found in
insect cuticle. They were designated as a tyrosinase-type phenoloxidase, laccase-
type phenoloxidase (or laccase) and granular phenoloxidase. There had been a long
discussion on the roles and classification of these cuticular enzymes (Ashida and
Yamazaki 1990; Barrett 1991). Since they all have activity to oxidize o-diphenols,
their involvement in cuticle pigmentation and hardening during development has
been one of the important areas of investigation in insect cuticle physiology. It was
shown that tyrosinase-type phenoloxidase is synthesized in the hemocytes, then
secreted into the hemolymph, and finally transported into the cuticle (Ashida and
Brey 1995; Asano and Ashida 2001). Granular-PO (GPO) was originally purified
from granules in the larval cuticle of M. sexta by using a preparative electrophoretic
gel in the presence of sodium dodecyl sulfate (see Sect. 6.4; Hiruma and Riddiford
1988). As observed in several other insect species, cuticular melanin was developed
within premelanin granules deposited into the outer procuticle (Kayser-Wegmann
1976; Curtis et al. 1984). The GPO is thought to be responsible for production of
melanin in such structures, but the gene for the granular enzyme has not been
identified. To show the involvement of proPOs and laccase-like MCOs in cuticle
formation during development, the phenotypes resulting from knockdown of these
genes by RNAi were compared in T. castaneum (Arakane et al. 2005). dsRNAs for
proPO genes (both of TcTyr1 and TcTyr2), laccase 1 (TcLac1) and TcLac2 were
injected. In contrast to the result that knockdown of TcLac2 induced severe defects
in pigmentation and abnormal adult shapes, no visible phenotypes were observed
when TcTyr1, TcTyr2 and TcLac1 were knocked down. This result strongly suggests
that the gene for the tyrosinase-type protein, proPO, is not involved in the process
of cuticle pigmentation and hardening during development.
Since the study of laccase 2 in T. castaneum, the RNAi method has been adopted
for use in other species from multiple orders to study the involvement of laccase 2
genes in cuticle formation (Niu et al. 2008; Elias-Neto et al. 2010; Futahashi et al.
2010; Ye et al. 2015). In all cases, pigmentation was suppressed. In several cases,
the cuticle showed an abnormal shape and became mechanically weak (Arakane
et al. 2005; Ye et al. 2015). RNAi for TcLac2 also reduced egg hatching rates at low
humidity, demonstrating that the enzyme is crucial for sclerotization of the serosal
cuticle and for embryonic desiccation resistance (Jacobs et al. 2015). In the larval
integument of B. mori, the spatial patterns of BmLac2 expression exhibit a close
correlation with those of pigmentation. The genes for substrate synthesis (TH, DDC,
tan and ebony in Fig. 6.1) are also expressed strongly in the areas of black and
reddish pigmentation (Futahashi and Fujiwara 2005, 2007; Futahashi et al. 2010).
In the RNAi study on TcLac2 from T. castaneum (Arakane et al. 2005), the authors
also described its characteristic gene structure, indicating the formation of two
splice variants, A- and B-type TcLac2. The protein products from the two variants
have the same N-terminal 491 amino acids, but the remaining C-terminal portion is
encoded by a distinct set of exons. The C-terminal variable region includes copper-
binding sites that are indispensable for oxygen binding inside the active center. It is
assumed that the two isoforms have different enzymatic properties for versatility in
functions. The presence of A-type and B-type isoforms is also found in An. gambiae
(Gorman et al. 2008) and at least three variants are found in D. melanogaster
(Flybase, http://flybase.org/; Asano et al. in preparation). Although it has not been
proven experimentally, the gene structure of BmLac2 implies that the A- and B-types
can be produced by similar splicing patterns (Yatsu and Asano 2009). The expres-
sions of A- and B-type variants were compared by PCR in T. castaneum and An.
gambiae. In each case, the temporal peak of expression is slightly different between
the two isoforms (Arakane et al. 2005; Gorman et al. 2008), indicating that each has
a unique function related to a specific timing. The knockdown of each isoform in T.
castaneum led to lethal phenotypes, but the deformation of the cuticle was more
severe in the knockdown of the A-type isoform.
Amino acid sequence analysis was performed for a laccase-like enzyme that was
purified from the newly ecdyzed pupae of B. mori (Yatsu and Asano 2009). The
procedure for the purification was a modified version of the previous report by
Yamazaki (1972). Like the previous study, trypsin was used for solubilization of
laccase activity. Since laccase is attached to the cuticle matrix very tightly, proteases
or denaturing regents are needed to break down the cuticle structure anchoring the
laccase protein. The purity was increased from three bands in the previous study to
only a single band (70 kDa) by analysis with SDS-PAGE. The N-terminal sequence
of the purified enzyme was NPALS that corresponds to Asn147-Ser151 of the full-
length putative polypeptide deduced from the cDNA sequence of BmLac2 (Fig.
6.4). The mass spectrometric identification of tryptic fragments from the purified
enzyme (trypsin-solubilized B. mori laccase 2, Bm-tLac2) failed to identify pep-
tides corresponding to 146 amino acids from the N-terminal methionine, suggesting
that the purified enzyme had lost the N-terminal portion during treatment with tryp-
sin. A similar result was reported in the analysis Lac2 from M. sexta. The full-length
and N-terminally truncated (Δ106) recombinant proteins exhibit similar catalytic
parameters when NADA is used as substrate, indicating that the presence of the
N-terminal portion does not have a significant influence on the enzymatic activity
(Dittmer et al. 2009). After these studies, the molecular properties of the recombi-
nant laccase 2 proteins of T. castaneum and An. gambiae were determined (Gorman
et al. 2012). They exhibit the same pattern of Km values with dopamine and NADA
being the more preferred substrates than DOPA and NBAD, respectively. The pH
optima of enzyme activities are also weakly acidic and the A-type isoforms exhibit
higher pH optima for DOPA and dopamine than the B-type isoforms. The four
recombinant proteins show similar kinetic parameters, but a notable difference was
seen in the case of TcLac2B with its kcat values much larger than the values deter-
mined for TcLac2A. The variation in pH optima and substrate preference in the two
isoforms may reflect the environment in which each isoform is localized.
Fig. 6.4 Schematic domain structures of 3dMCOs from invertebrates. Two proteins from insects
and three proteins from other invertebrate species are shown. Structural characteristics are
highlighted including the signal sequence, catalytic domain, cysteine-rich region, GPI anchor site,
von-Willebrand factor domains and copper-binding sites. Numbers indicate length of proteins (in
numbers of amino acids)
Since the early studies, there have been observations that imply an active
regulation of laccase function during the process of cuticle construction in D. virillis
(Yamazaki 1969) and the Australian sheep blowfly, Lucilia cuprina (Barrett 1987b).
In B. mori, little laccase activity was detected in the cuticle of newly ecdysed pupa,
but the activity increased to a maximum at 4 h after pupal ecdysis (Yamazaki 1972).
A similar result was obtained by in situ staining of the pupal cuticle with dopamine
(Yatsu and Asano 2009). It had been suggested that laccase is synthesized as an
inactive precursor and accumulates in the pupal cuticle before ecdysis. After pupal
ecdysis, the precursor may be activated through processing or interactions with
other molecule(s). A precursor-like protein was extracted from the cuticle of newly
ecdyzed pupae of B. mori by treatment with α-chymotrypsin. When examined by
activity staining after native PAGE, only a faint activity band was detected, but after
in vitro treatment with trypsin, an increased activity was detected (Ashida and
Yamazaki 1990; Asano et al. 2014). The purified α-chymotrypsin-solubilized
BmLac2 (Bm-cLac2) protein was shown to be an active enzyme, but the specific
activity was increased 17-fold after treatment with trypsin. During that treatment,
peptide bonds in the C-terminal region (Lys677-Gln678 and Arg674-His675) were
cleaved. Although it is not an inactive precursor, Bm-cLac2 appears to preserve the
character of the hypothetical inactive-precursor in that it has the potential to become
substantially more active. For the precise control of a successful completion of the
entire ecdysial process, it is reasonable to utilize multiple systems to regulate
laccase 2 activity. It is hypothesized that laccase 2 can become a much more active
enzyme by some activating factors through proteolytic processing or other modes of
interactions (Asano et al. 2014).
6.2.8 Dopachrome Conversion Enzyme (Yellow)
The yellow gene, which was named for a yellowish body color in a loss of function
phenotype of D. melanogaster (Nash and Yarkin 1974), appears to be one of the
most rapidly evolving gene families that generates functionally diverse paralogs.
This family includes the major royal jelly proteins (MRJPs) of the honeybee, Apis
mellifera, which are the most abundant proteins found in royal jelly (Schmitzova
et al. 1998; Albert and Klaudiny 2004). The yellow genes encoding MRJPs and
MRJP-like (MRJPL) proteins have been identified not only in honeybees but also in
several other hymenopteran species. Those MRJP and MRJPL genes are generally
arranged in a cluster (except for a single copy of MRJPL in some species) of closely
linked genes, which is located between yellow-e3 and yellow-h (most common gene
order is yellow-g, −g2, −e, −e3, MRJPs or MRJPLs, −h) in their genomes (Ferguson
et al. 2011a; Kupke et al. 2012; Buttstedt et al. 2014). Although the MRJPs or
MRJPLs locus is highly conserved, recent studies including phylogenetic analysis
suggest that the gene clades in A. mellifera, the jewel wasp, Nasonia vitripennis, and
the Argentine ant, Linepithema humile, have independently evolved in their lineage
(Fig. 6.5; Werren et al. 2010; Smith et al. 2011; Buttstedt et al. 2014). MRJP proteins
were detected in a variety of tissues including brain, venom glands and larval
hemolymph and in different developmental stages in A. mellifera (Kucharski et al.
1998; Schmitzova et al. 1998; Drapeau et al. 2006; Peiren et al. 2008; Randolt et al.
2008; Hojo et al. 2010), suggesting that MRJPs have other physiological functions
in addition to a nutritional role due to their high content of essential amino acids
(Table 6.3).
Yellow and MRJP/MRJPL comprise a gene family in insects. This gene family
of the melanin pathway appears to be insect specific and deviates from the pathway
common to other animals. The number of genes in insects, whose genomes are well
characterized, is in the range of 10–26 in species such as D. melanogaster (Maleszka
and Kucharski 2000; Drapeau 2001), T. castaneum (Arakane et al. 2010), B. mori
(Xia et al. 2006), P. xuthus (Futahashi et al. 2012), A. mellifera (Drapeau et al.
2006) and N. vitripennis (Werren et al. 2010). The encoded Yellow and MRJP/
MRJPL proteins have been divided into at least ten subgroups based on sequence
similarity and phylogenetic analysis (Fig. 6.5; Ferguson et al. 2011a). Although a
large number of yellow and MRJP/MRJPL genes has been identified in different
insect species, the physiological function(s) of most of these genes has not yet been
determined.
Fig. 6.5 Phylogenetic analysis of Yellow and Yellow-like proteins in insects. ClustalW software
was used to perform multiple sequence alignments prior to phylogenetic tree analysis. The
phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-Joining method
(Tamura et al. 2013). Numbers by each branch indicate results of bootstrap analysis of 5000
replications. See Table 6.3 for the accession numbers of protein sequences used in this analysis
In the cuticle tanning pathway, a yellow gene encodes dopachrome conversion

enzyme (DCE) that converts dopachrome and dopaminechrome to dihydroxyindole
(DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), respectively, during
melanin biosynthesis (Fig. 6.2). DCE accelerates melanin synthesis in insects such
as D. melanogaster, B. mori, P. xuthus and T. castaneum (Wittkopp et al. 2002b;
Futahashi and Fujiwara 2007; Futahashi et al. 2008a; Arakane et al. 2010; Ito et al.
2010). The Yellow protein is critical for cuticle pigmentation in several species. In
D. melanogaster, for instance, yellow (DmY-y) is required for black melanin
production, because mutation of DmY-y leads to the light brown/yellowish cuticle
and localization of DmY-y protein is correlated with black pigmentation patterns in
adult cuticle (Wittkopp et al. 2002a, b, 2003a, 2009; Gompel et al. 2005). Riedel
Table 6.3 Accession numbers of insect Yellow and Yellow-like proteins used for phylogenetic
analysis
Species Abbreviation Protein Accession number
Tribolium TcY-b Yellow-b ACY71055
castaneum TcY-c Yellow-c ACY71056
TcY-e Yellow-e ACY71058
TcY-e3 Yellow-e3 ACY71057
TcY-f Yellow-f ACY71059
TcY-g Yellow-g ACY71060
TcY-g2 Yellow-g2 ACY71061
TcY-h Yellow-h ACY71062
TcY-y Yellow-y ACY71063
TcY-1 Yellow-1 ACY71064
Drosophila DmY-b Yellow-b NP_523586
melanogaster DmY-c Yellow-c NP_523570
DmY-d Yellow-d NP_523820
DmY-d2 Yellow-d2 NP_611788
DmY-e2 Yellow-e2 NP_650289
DmY-e3 Yellow-e3 NP_650288
DmY-e Yellow-e NP_524344
DmY-f Yellow-f NP_524335
DmY-f2 Yellow-f2 NP_650247
DmY-g Yellow-g NP_523888
DmY-g2 Yellow-g2 NP_647710
DmY-h Yellow-h NP_651912
DmY-y Yellow-y NP_476792
DmY-k Yellow-k NP_648772
Bombyx mori BmY-b Yellow-b BGIBMGA014224-TA
BmY-c Yellow-c ABC96696
BmY-d Yellow-d ABC96694
BmY-e Yellow-e BAI39592
BmY-f3 Yellow-f3 NP_001037424
BmY-f4 Yellow-f4 NP_001037428
BmY-h2 Yellow-h2 BGIBMGA007255-TA
BmY-h3 Yellow-h3 BGIBMGA007256-TA
BmY-y Yellow-y BAH11146
BmY-x Yellow-x NP_001037430
(continued)

Papilio xuthus PxY-c Yellow-c BAM18241
PxY-d Yellow-d BAM18620
PxY-e Yellow-e BAM17806
PxY-f3 Yellow-f3 BAM18870
PxY-f4 Yellow-f4 BAM18225
PxY-h2 Yellow-h2 BAM17969
PxY-h3 Yellow-h3 BAM17970
PxY-y Yellow-y BAF73474
PxY-x Yellow-x BAM18176
Apis melifera AmY-b Yellow-b XP_006569902
AmY-e3 Yellow-e3 ABB82366
AmY-e Yellow-e XP_003249426
AmY-f Yellow-f NP_001011635
AmY-g Yellow-g XP_396824
AmY-g2 Yellow-g2 XP_006559006
AmY-h Yellow-h ABB81847
AmY-y Yellow-y NP_001091693
AmY-x1 Yellow-x1 XP_006565008
AmY-x2 Yellow-x2 XP_001122824
AmMRJP1 Major Royal Jelly NP_001011579
Protein (MRJP) 1
AmMRJP2 MRJP2 NP_001011580
(continued)

Nasonia NvY-b Yellow-b NP_001154989
vitripennis NvY-e3 Yellow-e3 NP_001154982
NvY-e Yellow-e NP_001154985
NvY-f Yellow-f NP_001154968
NvY-g2a Yellow-g2a XP_001603544
NvY-g2b Yellow-g2b NP_001154986
NvY-g2c Yellow-g2c XP_001603573
NvY-h Yellow-h NP_001153394
NvY-y Yellow-y NP_001154977
NvY-x1a Yellow-x1a XP_001601771
NvY-x1b Yellow-x1b NP_001155026
NvY-x1c Yellow-x1c NP_001154990
NvY-x1e Yellow-x1e XP_001607252
NvY-x2 Yellow-x2 XP_001601022
NvMRJP1 MRJP1 XP_001603404
NvMRJP2 MRJP2 NP_001155025
Acyrthosiphon ApY-d5 Yellow-d5 XP_001942700
pisum ApY-d6 Yellow-d6 XP_001942648
ApY-e Yellow-e XP_001948479
ApY-g2 Yellow-g2 XP_001945004
ApY-g4 Yellow-g4 XP_001944949
ApY-y Yellow-y NP_001165848
et al. (2011) demonstrated that overexpression of DmY-y altered coloration of the

adult wing cuticle from brown to black. In addition, the multi-ligand endocytic
receptor Megalin (Mgl) plays a role in promoting internalization/endocytosis of
DmY-y. Loss of function of Mgl caused excess DmY-y, resulting in a black
pigmented wing similar to that seen in the adult in which DmY-y is overexpressed.
This result suggests that, in addition to the spatial expression, DmY-y function is
regulated by a cellular process such as an endocytosis for cuticle coloration of D.
melanogaster. Further studies are needed to know whether this is the case in other
insect species.
Like DmY-y, Yellow is also important for body pigmentation in lepidopteran
species. Expression of yellow in the swallowtail species P. xuthus (PxY-y), P. mach-
aon (PmY-y) and P. polytes (PpY-y) is correlated with black body markings of their
larval cuticles (Futahashi and Fujiwara 2007; Futahashi et al. 2010; Shirataki et al.
2010). Futahashi et al. (2012) utilized microarray analysis to screen for marking-
specific genes using six markings at 11 different stages of P. xuthus larvae and all
of the yellow gene family members were identified except for yellow-b. Interestingly,
yellow-h3 (PxY-h3) exhibited an expression pattern of development very similar to
that of PxY-y. In addition, PxY-h3 was expressed in the black pigmented markings,
indicating that, like PxY-y, it also is involved in marking-specific melanin synthesis
in P. xuthus. In B. mori, yellow-y (BmY-y) and yellow-e (BmY-e) had been character-
ized as genes responsible for the body color mutants, ch (chocolate) and bts (brown
head and tail spot), respectively (Futahashi et al. 2008b; Ito et al. 2010). The larval
body color of the ch mutant strain is a reddish brown instead of a normal black
color, whereas the bts mutant strain exhibited a reddish brown larval head and anal
plates instead of the white coloration found in the wild-type strain. These results
suggested that both BmY-y and BmY-e play critical roles in larval pigmentation of
B. mori.
In T. castaneum, 14 yellow genes have been identified and their different expres-
sion patterns indicated that yellow genes have diverse functions (Arakane et al.
2010). Functional importance of yellow in cuticle pigmentation has been identified
in several insect species as described above. Interestingly, knockdown of the yellow
(TcY-y) by RNAi had no effect on body pigmentation in the larva, pupa and adult
except for black pigmentation in the pterostigma of the hindwing (Fig. 6.2g; Arakane
et al. 2010), suggesting that TcY-y is not critical for T. castaneum body wall cuticle
pigmentation. Yellow-f (DmY-f) and -f2 (DmY-f2) in D. melanogaster showed a
DCE activity required for melanin synthesis (Han et al. 2002). However, unlike
PxY-y and PxY-h3, yellow-f3 (PxY-f3) and -f4 (PxY-f4), which are homologs of DmY-
f/-f2 in P. xuthus, were not up-regulated in the larval black markings (Futahashi
et al. 2012). RNAi for T. castaneum homolog of DmY-f/-f2 (TcY-f) had no effect on
body pigmentation of the larva, pupa and pharate adult. The resulting pharate adults,
however, were unable to shed their exuviae and died entrapped in their old pupal
cuticle (Arakane et al. 2010). These results suggested that Yellow-f-related genes
might have other functions in addition to melanin production in P. xuthus and T.
castaneum.
Recently, a novel anti-dehydration function of yellow-e (TcY-e) in T. castaneum
has been reported (Noh et al. 2015a). RNAi for TcY-e had no effect on larval, pupal
and pharate adult cuticle pigmentation. However, the resulting adults died shortly
after eclosion due to dehydration, and the lethality was prevented by high humidity.
The body color of the high humidity-rescued adults, like that observed in the B. mori
bts mutant, was significantly darker than that of control adults (Fig. 6.2f), suggest-
ing that TcY-e plays a role not only in body pigmentation but also has a vital water-
proofing function in T. castaneum adults. In contrast to loss of function of yellow,
which causes a lighter body color in many insect species, depletion of yellow-e
function results in a darker body pigmentation in both B. mori and T. castaneum.
The most prominent function of the yellow gene is in the production of black pig-
ment in a variety of insects. However, the function of the other members of the fam-
ily is largely obscure. Further study is required to understand the function of the
yellow paralogs across different insect species including the molecular mechanisms
for the cause of the dark body pigmentation in yellow-e deficient insects.
6.2.9 Structural Cuticle Proteins (CPs)
In arthropods, structural cuticular proteins (CPs) play important roles in determining

the diverse physical properties of the cuticle depending on developmental stages as
well as different body regions as a result of interactions with other CPs and the
structural biopolymer chitin (Neville 1993). Recent studies and the availability of
fully sequenced and annotated genomes of several insect species such as A. mellifera
(Honeybee Genome Sequencing Consortium. 2006), D. melanogaster (Karouzou
et al. 2007), T. castaneum (Dittmer et al. 2012; Tribolium Genome Sequencing
Consortium. 2008), An. gambiae (Cornman et al. 2008), B. mori (Futahashi et al.
2008a) and N. vitripennis (Werren et al. 2010) indicate that there are large numbers
of genes encoding CP-like proteins in their genomes (see Chap. 1 in this book for
details about CPs). Indeed, more than 200 putative CP genes have been identified in
D. melanogaster and An. gambiae (Ioannidou et al. 2014), and these genes comprise
~2 % of the predicted protein-coding genes in the latter species.
CPs have been classified into thirteen distinct families as defined by unique
amino acid sequence motifs characteristic of each of these families (Willis 2010;
Willis et al. 2012; Ioannidou et al. 2014). 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 (Rebers and Riddiford 1988). The
R&R motif contains a chitin-binding domain (chitin-bind-4 domain, PF00379 in the
Pfam database) that apparently helps to coordinate the interactions between chitin
fibers and the proteinaceous matrix (Rebers and Willis 2001; Togawa et al. 2004,
2007; Qin et al. 2009). CPR proteins containing the RR-1 motif have been found
primarily in soft and flexible cuticle, whereas the proteins containing the RR-2
motif have been found mostly in hard and rigid cuticle. The third class of CPR
proteins, RR-3, has been identified in only a few insect species and is a very minor
group whose distinguishable features have not yet been well defined (Andersen
2000).
During cuticle tanning, some of CPs are cross-linked by quinones and/or quinone
methides produced by laccase 2-mediated oxidation of N-acylcatechols (Hopkins
and Kramer 1992; Arakane et al. 2005; Andersen 2008; Mun et al. 2015). This vital
process together with dehydration occurs during each stage of development and the
expression of specific CPs appears to be required for formation of diverse cuticles
in different regions of the insect’s body and at different developmental stages so that
an appropriate combination of physical and morphological features can provide
structural support, mechanical protection and mobility. However, little is known
about the functional importance of individual insect CPs in the morphogenesis and
mechanical properties of the cuticle.
Four major CPs, TcCPR27, TcCPR18, TcCPR4 and TcCP30, were identified in
protein extracts of elytra from T. castaneum adults (Arakane et al. 2012; Dittmer
et al. 2012; Mun et al. 2015; Noh et al. 2015b). All of these CPs are abundant in
rigid cuticles including the dorsal elytron, pronotum, ventral abdomen and leg,
while they are absent or very minor in soft and flexible cuticles such as the ventral
elytron, hindwing and dorsal abdomen of the adult. TcCPR27 and TcCPR18 are
members of the RR-2 group of the CPR family, and are localized in both the
chitinous horizontal laminae and vertical pore canals in the procuticle of rigid adult
cuticle (Noh et al. 2014). RNAi for TcCPR27 or TcCPR18 genes caused a
disorganized laminar architecture and amorphous pore canal fibers (PCFs), which
led to short, wrinkled and weakened elytra (Arakane et al. 2012; Noh et al. 2014).
TcCPR4, which is more highly extractable from the elytra of TcCPR27-deficient
adults than are other CPs, contains an RR-1 motif (Noh et al. 2015b). TEM
immunogold labeling revealed that the TcCPR4 protein is predominantly localized
in the pore canals and in the vicinity of the apical plasma membrane protrusions of
the procuticle. However, depletion of TcCPR27 caused mislocalization of TcCPR4.
In the TcCPR27-deficient elytra, the TcCPR4 protein was distributed over the entire
procuticle including the horizontal laminae, suggesting that the presence of
TcCPR27 protein is critical for the specific localization of TcCPR4 protein. Loss of
function of TcCPR4 produced by RNAi caused an abnormal shape of the pore
canals with amorphous PCFs in their lumen, indicating that TcCPR4 is important
for determining the morphology and ultrastructure of PCFs and pore canals in rigid
cuticle.
Unlike TcCPR27, TcCPR18 and TcCPR4, the mature TcCP30 protein has a low
complexity amino acid sequence with a rather unique amino acid composition (36 %
Glu, 21 %, His, 19 %, Arg and 16 % Gly), and lacks an R&R consensus motif (Mun
et al. 2015). The function of TcCP30 is critical for adult eclosion of T. castaneum
probably because it undergoes cross-linking during cuticle maturation. Western
blotting analysis of protein extracts from the elytra and pronotum revealed that
TcCP30 becomes cross-linked to TcCPR27 and TcCPR18, but not to TcCPR4, by
the action of laccase 2 (Mun et al. 2015). Because TcCP30, TcCPR27 and TcCPR18
have a high histidine content (10–21 %), whereas TcCPR4 has a relatively low
content (3 %), this result appears to be consistent with the hypothesis that histidine
residues of CPs most likely participate in quinone- and/or quinone methide-mediated
CP cross-linking (Kramer et al. 2001). All of these results indicate that the unique
localization and cross-linking of specific CPs are important for morphology and
ultrastructure of the exoskeleton (Fig. 6.6). Genes encoding CPs comprise one of
the largest families of insect genes so that future studies of the functional importance
in cuticle morphogenesis of many other CPs, particularly ones belonging to different
subfamilies, is of great interest.
6.3 Interactions and Functions of Pigments in Insect Ecology
The surfaces of insects show various types of colorations, which include structural
colors and colors with versatile pigments. The structural colors are the results of
optical effects by fine structures of cuticle surfaces (Seago et al. 2009). In addition,
insect cuticles contain various types of substances that are responsible for
Fig. 6.6 Proposed cuticular proteins cross-linking in rigid cuticle of T. castaneum. Rigid adult
cuticle is composed of three functional layers, envelope (EV), epicuticle (EP) and procuticle
(PRO) in the 5 days-old adult of T. castaneum. Chitin and cuticular proteins are major components
of a large number of horizontal laminae and vertical pore canal fibers in the procuticle. Major
cuticular proteins, TcCPR27, TcCPR18 and TcCP30, are localized in both laminae and pore canal
fibers, whereas TcCPR4 is predominantly localized in pore canal fibers (Mun et al. 2015; Noh
et al. 2014, 2015b). TcCP30 undergoes cross-linking with TcCPR27 and TcCPR18, but not to
TcCPR4 in rigid cuticle (Mun et al. 2015). Pigments are primarily localized in the procuticle
characteristic colors. Sometimes, there are correlations between color (pigment)

patterns and the local shape of cuticle. For example, the Junonia butterfly wing
eyespot pattern correlates with the thickness pattern of the underlying cuticle (Taira
and Otaki 2016). With pigments, insects can create a variety of body color patterns
that contribute to increased survival rates through camouflage, warning or mimicry
and to accelerated sexual selection. The biological significance of body colors
produced through tyrosine metabolism has been investigated as one major research
area in many insect species. This section focuses mainly on the changes in the extent
of melanization that are associated with environmental conditions.
During development, insects often change their body colors for better adaptation
to the surroundings. As one example, the molecular mechanisms of body color tran-
sition are well described in P. xuthus (Futahashi and Fujiwara 2005, 2008). Until the
fourth instar, the larvae show a pattern with white and brown colors that is thought
to mimic the fecal material of birds. In contrast, during the molt to the fifth (last)
instar, the pattern drastically changes to a greenish coloration that is cryptic for
leaves. For this transition of body color pattern, the concentration of juvenile hor-
mones (JH) is the critical factor (see Sect. 6.4). This transition in P. xuthus is an
example of events that occur during the normal developmental process. Similarly,
polyphenic phenotypes occur through changes in gene expressions that are regu-
lated by hormones (JH, ecdysteroid, peptidic factors) through modulations in the

status of epigenetic modifications and metabolic pathways. The body color pattern
is one polyphenic trait that can be modified by environmental cues. Locusts have
two phases, a “solitaria phase” in low density of individuals and a “gregaria phase”
under the opposite situation, each of which is characterized by a specific metabo-
lism, behavior and body appearances. In the transition from solitary to gregarial
phase, the greenish body is changed to blackish. By HPLC isolation and bioassay
experiments with the albino mutant of L. migratoria, the peptide corazonin was
identified from both L. migratoria and S. gregaria as the hormone that induces mel-
anin synthesis (Tawfik et al. 1999; Tanaka 2000; Predel et al. 2007). Corazonin has
an N-terminal pyroglutamate and C-terminal amidation, and the sequence is homol-
ogous to the human melanocyte-stimulating hormone α-MSH. Corazonins from the
locust have a histidine residue at the seventh position instead of an arginine in cora-
zonin from the cockroach, P. americana, and the peptide was identified as a heart-
stimulating hormone in P. americana (Veenstra 1989, 1991). Corazonin induces not
only melanogenesis, but also morphological changes (Tanaka et al. 2002; Maeno
et al. 2004). It has been shown in S. gregaria that in a crowded situation, the signal
of mechanoreception on the hind legs is transduced to the central nervous system
(CNS). Then neuromodulation of the CNS leads to behavioral changes from solitary
to the gregarious type. Either mechanoreception at the hind legs or a combination of
sight and odor cues induce a pulse of serotonin in the metathoracic ganglion, and
this is both necessary and sufficient for induction of behavioral changes for
gregarization in S. gregaria (Simpson et al. 2011). The eggs laid by gregarious
females have a tendency to be larger than those laid by solitary females and the
proportion of “green hatching” from the larger eggs is lower (Tanaka and Maeno
2010).
Temperature appears to cause adaptive changes in the coloration pattern. The
larval body of the five-spotted hawkmoth, Manduca quinquemaculata, shows two
temperature-dependent phenotypes. Larvae developed at 20 °C have a blackened
color, but those developed at 28 °C have a greenish color (Hudson 1966). The dark
body color can be adaptive to obtain heat by absorbing sunlight in a low temperature
condition, although the black coloration is thought to be in a trade-off relationship
with the cryptic green color in leaves. In the related species, M. sexta, the black
mutant shows similar characteristics. At normal temperature (20–28 °C) the body
color is black, and low JH titer is responsible for this phenotype. In contrast, heat
shock during the sensitive period in the fourth instar elevates the JH titer and
suppresses DDC expression during the molt. The body color of the next instar is
changed to green (Suzuki and Nijhout 2006). Such experiments to decipher the
effect of temperature shifts have been performed since the nineteenth century. In
many cases, a high-temperature shock induced phenotypes found in warm climates,
and vice versa (Merrifield 1890, 1893). In a nymphalid butterfly, Junonia orithya, a
cold shock during the pupal period shows polyphenic wing color patterns that are
induced by a cold-shock hormone (Mahdi et al. 2010). Although such a correlation
has been elusive, the cold-shocked individuals show an increase in dopamine
content and a tendency to produce a darker phenotype. In D. melanogaster, growth
at a lower temperature induces a darker abdomen in adult females (David et al.

1990), which is associated with modulations of TH and ebony expressions (Gibert
et al. 2007). This observation is consistent with the geographic distributions of
darker variations (David et al. 1985; Gibert et al. 1996, 1998; Takahashi and Takano-
Shimizu 2011; Telonis-Scott et al. 2011), indicating that this plasticity is an adaptive
trait. The relationship between high latitudes (low temperature and weak sunlight)
and darker body color has been reported in many cases (reviewed by True 2003;
Rajpurohit and Nedved 2013; Takahashi 2013). The recent global warming trend
has affected greatly the distribution of European insects (Zeuss et al. 2014). Based
on the data obtained from a study of 473 European butterflies and dragonflies, it was
found that dark and light colored insects are associated with cool and warm climates,
respectively, and that the average darkness of the insects has decreased during the
last century.
6.4 Hormonal Regulation of Cuticle Tanning
How does hormonal regulation lead to variations in pigmentation of the insect exo-
skeleton? Pigmentation development involves activating agents such as hormones
that regulate the process, positioning agents that generate pigments in space and
time, and the biochemical synthesis of pigments (Wittkopp and Beldade 2009).
Genes in these steps are referred to as “regulating”, “patterning” and “effector”
genes, respectively. Regulating genes modulate patterning genes that determine the
distribution of pigments by directly or indirectly activating expression of effector
genes that encode the enzymes and co-factors required for pigment biosynthesis.
There are two main classes of insect hormones, the hormones produced by epi-
thelial glands belonging to the ecdysteroids and juvenile hormones (JH) and also the
neuropeptide hormones produced by neurosecretory cells. A working hypothesis is
that the pigment biochemical module is regulated primarily by ecdysteroids such as
20-hydroxyecdysone (20HE) and JH as well as transcription factors expressed in a
marking-specific pattern. Neuropeptide hormones such as bursicon also play regu-
latory roles in pigment metabolism.
Cuticular pigmentation in P. xuthus is caused by exposure to ecdysteroid and its
subsequent removal (Futahashi and Fujiwara 2007). For instance, the larval marking
eyespot pigmentation and differences in pigmentation timing appear to be regulated
by ecdysteroid-inducible transcription factors that modulate melanin-synthesis
genes. In this species, stage-specific larval marking color patterns are determined by
a two-phase melanin-related gene prepatterning process (Futahashi et al. 2010).
Two secreted protein genes, yellow-y and laccase 2, correlated with the black
markings are expressed in the middle of the molting period (phase 1). In contrast,
five epidermal proteins genes, TH, DDC, GTPCH I (GTP cyclohydrolase I), tan and
ebony, associated with black or reddish-brown markings are expressed in the latter
half of the molting period (phase 2). In addition, topical application of ecdysteroids
prevents the induction of TH, DDC and ebony expression as well as maintains
yellow expression in the later molting stage, suggesting that the genes expressed in
phase 1 require a high ecdysteroid titer, whereas the expression of the genes in
phase 2 are induced by 20HE removal. Similar spatial and temporal expression
profiles of most corresponding genes are observed in two other Papilio species with
two additional genes, bilin-binding protein (BBP) and a yellow-related gene (YRG),
whose combinations contribute larval blue, yellow and green colorations (Shirataki
et al. 2010).
Two transcription factors, spalt and ecdysteroid signal-related E75, are genes
expressed in the larval black eyespot markings in Papilio (Futahashi et al. 2012).
The former expression correlated with the cryptic wing pattern (black marking)
formation in several butterfly species, suggesting that it may be a positive regulator
for melanin synthesis in both larvae and adult butterflies. The expression pattern of
E75 induced by ecdysteroids (Hiruma and Riddiford 2009) is correlated with the
eyespot pigmentation pattern, which is similar to that of the yellow gene. In M.
sexta, DDC expression is regulated by E75 in response to exposure to ecdysteroids
followed by its removal (Hiruma and Riddiford 1990, 2009). In B. mori, tan and
laccase 2 genes, which are also marking-specific (Futahashi et al. 2010), are
apparently regulated by several ecdysteroid-inducible transcription factors including
ecdysone receptor, E75, hormone receptor 3 and ftz transcription factor 1 (Hiruma
and Riddiford 2009). These results suggested that E75 is a strong candidate mediator
of the hormone-dependent coordination of larval pigment pattern formation by
regulating several black marking genes. Transcription factors such as optomotor-
blind (omb) or bric a brac (bab) may also regulate patterning in insects (Wittkopp
et al. 2003b). Color patterns in wings of butterflies are complex and may be the
product of the co-option of developmental pathways, as exemplified by the eye
development gene optix, which is correlated with wing patterns of Heliconius
butterflies (Kronforst et al. 2012).
JH has been proposed to influence the expression pattern of 20HE-related genes
(Riddiford 2008), and it induces the expression of genes associated with the mimetic
pattern. In P. xuthus, a decrease in JH titer causes a switch to the cryptic pattern
(Futahashi and Fujiwara 2008). Until the end of third instar larva, a high JH titer is
maintained, but during the fourth instar larval stage, the titer declines. The low JH
titer is the cause of the greenish cryptic pattern in the next instar, because injection
of a JH analog (JH acid: JHA) at the early fourth instar larval stage suppresses the
transition of body color pattern. With the injection of JHA, the spatial patterns of
TH and DDC expressions in the cuticles of fourth instar larvae are maintained
during the molt, and the mimicking brownish color pattern is maintained in the
resulting fifth instar larvae. In addition to overall coloration, JH also regulates
exoskeletal structures and pigment distribution at specific markings. In general, the
hormonal condition and hormone sensitivity appear to be two of the main factors
responsible for interspecific differences in larval color patterns in lepidopteran
species such as the genus of Papilio (Shirataki et al. 2010).
The pupal color of the butterflies often shows dimorphic phenotypes, green and
brown. The occurrence of the colors is dependent on the surrounding situations at
the site for pupation. Optic signals from background color or tactile signals from the
surface of the pupation site (smooth or rough) have been studied as determinants for
pupal colors (Hazel and West 1979; Hiraga 2005). Seasonal factors (photoperiod,
temperature or humidity) can also have an influence on the pupal color determination
(Hazel and West 1979, 1983; Sims and Shapiro 1984; Smith 1978, 1980). In the
nymphalid butterfly, Inachis io, the browning of the pupa is controlled by the
neuropeptide, pupal melanization-reducing factor (PMRF), that is released from the
brain into hemolymph (Bückmann and Maisch 1987). In P. xuthus, the pupal color
is controlled by pupal cuticle melanizing hormone (PCMH) and orange pupa
inducing factor (OPIF; Yamanaka et al. 1999, 2004, 2006). Blackening of body
color occurs in larvae of the armyworm, P. separata. In crowded conditions,
“melanization and reddish coloration hormone” (MRCH) known for its FxPRL-
amide structure at the C-terminus is released into the hemolymph to increase
melanization of the cuticle. MRCH is homologous to the insulin-like growth factor
(Matsumoto et al. 1981, 1986). In P. separata other physical stresses (cold shock or
continuous mechanical stimuli) or parasitic wasp injection elevates the level of
another type of peptide that shares sequence similarity with the epidermal growth
factor (Hayakawa et al. 1995; Ohnishi 1954). This factor, growth-blocking peptide
(GBP), is responsible for suppression of the increase in JH esterase levels after
parasitization (Hayakawa 1991). GBP directly acts on epidermal cells to induce an
elevation of cytoplasmic calcium ion concentration, which increases the levels of
TH and DDC expression (Ninomiya and Hayakawa 2007).
At the enzyme level, the epidermal synthesis of a granular phenoloxidase (GPO)
that catalyzes cuticular melanization of M. sexta larvae is inhibited by JH (Hiruma
and Riddiford 1988). The absence of JH at the time of head capsule slippage during
the last larval molt causes deposition of premelanin granules containing an inactive
prophenoloxidase into the outer regions of the newly forming endocuticle. The
decline of ecdysteroid titer allows activation of the proGPO and subsequent
melanization (Curtis et al. 1984). Neuropeptide hormones such as MRCH, eclosion
hormone, or extracts of pharate-larval subesophageal ganglia or corpora cardiaca-
corpora allata complexes did not accelerate melanization. Dopamine, the precursor
for melanin, is produced by the epidermis at the end of the molt, due to an increase
in DOPA decarboxylase (DDC), which catalyzes dopamine production from DOPA
(Hiruma and Riddiford 1993). The rise in DDC activity is controlled at the
transcriptional level and dependent on the changing ecdysteroid titer during the
molt as described above.
Bursicon (bur) known as “tanning hormone” is an insect heterodimeric neuro-
peptide hormone that is secreted from the central nervous system into the hemo-
lymph, which initiates cuticle tanning as well as wing expansion after adult eclosion.
Rickets (rk), the receptor for bur, is a member of the G protein-coupled receptor
superfamily (Honegger et al. 2008; Luo et al. 2005; Mendive et al. 2005). In D.
melanogaster larvae, Dmbur is expressed exclusively in crustacean cardioactive
peptide-expressing neurons (Luan et al. 2006; Luo et al. 2005; Park et al. 2003). D.
melanogaster mutants for Dmbur and the receptor mutant Dmrk showed deficiencies
in both tanning and wing expansion (Baker and Truman 2002; Dewey et al. 2004).
Eighty-seven genes in D. melanogaster were found to be sensitive to recombinant
bur stimulation (An et al. 2008). Thirty have no known function, but the others
encode proteins with diverse functions, including cell signaling, gene transcription,
DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism,
cytoskeleton formation, immune response and cell-adhesion. Huang et al. (2007)
reported that loss of function of Bmbur caused a defect in wing expansion but no
distinct tanning phenotype was observed in B. mori adults. Similarly, knockdown in
the expression of genes coding for Tcbur and/or its receptor Tcrk in T. castaneum by
injecting dsRNA into the pharate pupae caused a wrinkled elytra phenotype, but
showed no effect on cuticle tanning. However, when Bai and Palli (2010) injected
Tcrk dsRNA earlier in development (into young last instar larvae), pupal cuticle
tanning was affected. A reduction in the expression of laccase 2 in Tcrk RNAi
beetles suggested that Tcrk influences pupal cuticle tanning by regulating the
expression of laccase 2 in T. castaneum. However, post-translational modifications
such as phosphorylation may also play a role in regulation of cuticle tanning by the
bursicon receptor (Honegger et al. 2008). TH is transiently activated during tanning
by phosphorylation at a serine residue as a result of bursicon signaling (Davis et al.
2007). A novel bursicon-regulated gene in the housefly, Musca domestica (named
md13379 because it is homologous with CG13379 in D. melanogaster), encodes a
transcriptional regulator homologous to human ataxin-7-like3 and yeast sgf11, both
of which encode a novel subunit of the Spt-Ada-Gcn5-acetyltransferase (An et al.
2009). Proteins related to md13379 may play a role in regulating the expression of
bursicon-regulated genes involved in cuticle tanning.
6.5 Future Prospects and Concluding Remarks
Although tyrosine metabolism for insect cuticle tanning and the chemistry of pigment
synthesis in general are still not well understood, past studies have demonstrated sev-
eral physiologically and ecologically important roles in the tanning process. These
findings suggest that the potential for development of novel control agents targeting
insect cuticle tanning physiology. For example, after demonstration of specific gene
down-regulation in several insect species including crop pests by orally administered
dsRNA (Turner et al. 2006; Zhou et al. 2008; Tian et al. 2009; Walshe et al. 2009;
Chen et al. 2010; Huvenne and Smagghe 2010; Zhang et al. 2010; Li et al. 2011;
Bolognesi et al. 2012; Zhu et al. 2012; Miyata et al. 2014; Wan et al. 2014; Guo et al.
2015), RNAi based-pest management is now considered to be a promising new tech-
nology to control target pest insects with high species-specificity and environmentally
friendly attributes. In addition, transgenic plants expressing dsRNAs for target insect
genes impair growth and development (Baum et al. 2007; Mao et al. 2007; Pitino
et al. 2011; Zha et al. 2011; Zhu et al. 2012; Kumar et al. 2013; Zhang et al. 2015).
These results suggest that RNAi based-pest control could be a very useful alternative
to chemical pesticides for suppression of economically damaging pest insects. Loss
of function of many genes involved in the tyrosine-mediated cuticle tanning pathway
caused not only alterations in color but also resulted in high mortality as described in
Sect. 6.2, indicating that the pathway is vital for insect development, growth and sur-
vival, making the genes potential targets for controlling economically important pest
insects and animal/plant disease vectors.
The involvement of phenoloxidases in cuticle tanning is essential to insect sur-
vival (Dittmer and Kanost 2010) and laccases are potential phenoloxidase targets
for insect control agents. One of the first compounds developed to adversely affect
insect cuticle tanning is the sterically hindered phenol, 2,6-ditert-butyl-4-(2-
phenylpropan-2-yl)-phenol (MON-0585, Semensi and Sugumaran 1986). In MON-
0585-treated insects there was a reduced phenoloxidase activity as compared to
controls as well as a significant reduction in the level of covalently bound catechols
and malformations in the exoskeleton. Prasain et al. (2012) tested substituted
phenolic compounds as substrates or inhibitors of laccase and also examined their
effects on mosquito larval growth. Several compounds caused >90 % mortalities as
mosquito larvicides at nM-μM concentrations. The treated larvae started to separate
the larval cuticle, but they failed to shed it. Also there was very little synthesis of
new pupal cuticle by the treated larvae. Thus, some of the laccase-active compounds
affected the development of the cuticle, which is a target that is different from the
neurological targets of most currently utilized insecticides. However, the exact
target and mechanism of action of these modified phenolic compounds in vivo still
remain to be determined.
In biomimetic/entomomimetic science, an area of research involves the
development of novel biomimetic materials with unique physical properties similar
to those of the pigmented insect exoskeleton for use in biomedical or other
technological devices. For example, a pigmented cuticle structure of the highly
modified and tanned forewing elytron of beetles was investigated as a biomimetic
model and its application was described (Dai and Yang 2010; Lomakin et al. 2011;
Chen and Wu 2013; Chen et al. 2015a, b). Unlike other pigmented and rigid cuticles
in different body regions of adult beetles, the elytron composes of dual cuticular
layers (dorsal and ventral cuticles) connected by a large number of pillar-like fibrous
structures called trabeculae. The unique structure of the elytron, for instance, has
been applied as a model to develop novel integrated honeycomb structures that are
lightweight with a high mechanical strength (Chen et al. 2012, 2015b).
Although substantial progress in studies of insect cuticle tanning metabolism has
occurred in the past, there remain several unanswered questions that should be
addressed in future studies. What are the exact chemical structures of the insect
pigments? Structural models and templates have been offered for some of the
pigments including eumelanin and pheomelanin (Solano 2014), but the exact
structures have not been established. How do the pigments interact with other
components for assembly of the supramolecular extracellular matrix known as the
exoskeleton? How do the pigments interact covalently and non-covalently with
other components in the cuticle including proteins, chitin, lipids, minerals and
water? What is the contribution of the pigments to the mechanical properties of
exoskeletons? Substantial research designed to answer these questions remains to
be conducted in the future so that we will have a more complete understanding of
the chemistry, physiology and genetics of insect cuticle pigmentation and

sclerotization.
Acknowledgements 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), Bio-industry Technology Development
Program (111107011SB010), Ministry for Food, Agriculture, Forestry and Fisheries, and Basic
Science Research Program through the NRF funded by the Ministry of Education (NRF-
2015R1A6A3A04060323) to MYN.
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Tyrosine Metabolism For Insect Cuticle Pigmentation and Sclerotization

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Chapter 6

Tyrosine Metabolism for Insect Cuticle

Yasuyuki Arakane, Mi Young Noh, Tsunaki Asano, and Karl J. Kramer

Y. Arakane (*) • M.Y. Noh

© Springer International Publishing Switzerland 2016 165

6.1.1 Insect Cuticle Composition and Morphology

The insect exoskeleton or cuticle is composed of multiple functional layers includ-

6.1.2 Proposed Tyrosine-Mediated Cuticle Tanning Pathway

Despite a rather limited composition, cuticle has remarkably diverse mechanical

6.2 Functions of Key Enzymes/Proteins Involved in Tyrosine-

6.2.1 Tyrosine Hydroxylase (TH)

Fig. 6.2 Proposed cuticle tanning pathway in T. castaneum. DOPA, 3,4-dihydroxyphenylalanine;

oxidizing ortho-diphenols as well as hydroxylating monophenols such as tyrosine

Oncopeltus fasciatus KM247780 RNAi Loss of black Liu et al. (2014)

Table 6.1 (continued)

Table 6.1 (continued)

6.2.2 Dopa Decarboxylase (DDC)

Dopamine is a catecholamine involved in nervous systems of mammals and insects

Similarly, loss of function of DDC by RNAi causes a reduction in black pigmentation

6.2.3 Aspartate 1-Decarboxylase (ADC)

β-Alanine is involved in critical physiological events in insects. In cuticle tanning,

strains of T. castaneum exhibit decreased concentrations of both β-alanine and

6.2.4 Arylalkylamine N-Acetyltransferase (AANAT)

Arylalkylamine N-acetyltransferases (AANATs) belong to a large Gcn5-related

Klein 2007). AANAT activity is rate-limiting and it acetylates serotonin to form

6.2.5 NBAD Synthase (Ebony)

NBAD synthase can be defined with a more general name, β-alanylbiogenic

6.2.6 NBAD Hydrolase (Tan)

6.2.7 Laccase (Lac)

6.2.8 Dopachrome Conversion Enzyme (Yellow)

In the cuticle tanning pathway, a yellow gene encodes dopachrome conversion

Table 6.3 (continued)

Table 6.3 (continued)

et al. (2011) demonstrated that overexpression of DmY-y altered coloration of the

6.2.9 Structural Cuticle Proteins (CPs)

In arthropods, structural cuticular proteins (CPs) play important roles in determining

6.3 Interactions and Functions of Pigments in Insect Ecology

characteristic colors. Sometimes, there are correlations between color (pigment)

lated by hormones (JH, ecdysteroid, peptidic factors) through modulations in the

at a lower temperature induces a darker abdomen in adult females (David et al.

6.4 Hormonal Regulation of Cuticle Tanning

6.5 Future Prospects and Concluding Remarks

the chemistry, physiology and genetics of insect cuticle pigmentation and

Bolognesi R, Ramaseshadri P, Anderson J, Bachman P, Clinton W, Flannagan R, Ilagan O,

Dai F, Qiao L, Cao C, Liu X, Tong X, He S, Hu H, Zhang L, Wu S, Tan D, Xiang Z, Lu C (2015)

Finocchiaro L, Callebert J, Launay JM, Jallon JM (1988) Melatonin biosynthesis in Drosophila:

Sideri M, Tsakas S, Markoutsa E, Lampropoulou M, Marmaras VJ (2008) Innate immunity in

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