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Manuscript_c8f49414ce7f9886cc2c06a14d4a1221

Advance in Insect Physiology

Insect multicopper oxidase-2 (MCO2) and its roles in cuticle formation

Tsunaki Asano
Department of Biological Sciences, Tokyo Metropolitan University, Tokyo, Japan

Key words: cuticle, laccase; MCO2; diphenols; oxidation; terrestrialization

1. Introduction
2. Mechanisms of cuticle hardening in arthropods
2.1 Cuticle hardening in non-insect arthropods
2.2 Cuticle hardening in insects
3. Phenol-oxidizing enzymes in insect cuticle
3.1 Laccase-type enzymes
3.1.1 Overview of laccases and multi copper oxidases
3.1.2 Laccase-type enzymes in insect cuticles
3.1.3 Biochemical analyses of MCO2s
3.1.4 Phenotypic analyses of the genes for MCO2s
3.1.5 Programs for cuticle formation
3.2 Tyrosinase-type enzymes
3.2.1 Overview of tyrosinase-type enzyme in insects
3.2.2 Molecular identification of tyrosinase-type enzyme in insect cuticle
3.2.3 Evolution of type-3 protein in arthropods
4. Evolution of the genes for MCOs in arthropods, and possible contributions of
MCO2 in the success of insects as terrestrial animals.
4.1 Evolution of MCO2 in the history of arthropods’ evolution
4.2 MCOs in insects: MCO1s, MCO-related proteins and others
4.3 Possible advantages of MCO2-mediated system in colonization of land
4.4 Candidates of enzymes that substitute functions of MCO2
4.5 Adaptation to terrestrial environments by MCO2-mediated system and the
reason why insects are rare in marine ecosystems
5. Concluding remarks
6. Acknowledgement

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1. Introduction
Since the early periods of the studies suggesting the involvement of o-diphenols in the
process of insect cuticle formation, there had been studies on several types of
phenol-oxidizing enzymes obtained from cuticle samples. Among the cuticular enzymes,
it has been confirmed during the recent two decades that laccase-type enzyme is the
most important enzyme responsible for cuticle melanization and hardening in normal
developmental process. The name, “laccase” came from the enzyme that was originally
found from the cell sap of the Japanese “lacquer” tree. Since the finding of laccases
from plants or fungi during the late 19th century, their properties, structures, and
functions have been extensively investigated by many investigators. Currently, the
laccase is classified into a group of three domain multi copper oxidases (3dMCO). In
insects, molecular properties and functions of enzymes that have enzymological
properties similar to those of plant and fungal laccases have been studied for over 65
years, but it was relatively recent events, compared to those of other organisms, that the
gene for the laccase-type insect enzyme was identified. The laccase-type enzymes in
insect cuticle also belong to the members of 3dMCOs, and they are currently referred to
as laccase2 or MCO2 in insect science. Since the first demonstration by RNA
interference experiments to show that the gene for MCO2 has indispensable role in
cuticle formation, the study of MCO2 genes has become very active. This review aims
to summarize the recent studies on the functions of the laccase-type enzymes in insect
cuticle formation, and also introduces a new hypothesis regarding how the
MCO2-mediated system of cuticle hardening might have affected to the direction of
insects’ evolution or the success of insects as terrestrial animals.

2. Mechanisms of cuticle hardening in arthropods

2.1 Cuticle hardening in non-insect arthropods

Insects are classified into the phylum, Arthropoda, and are ordinarily characterized with
three pairs of the legs and flight ability by the wings. Like other arthropods, insects have
cuticles that cover the entire surface of insect body including trachea, foregut, and
hindgut. The cuticle serves as the exoskeleton for the mechanical support of body
shapes and movements and also behaves as a skin to divide inner and outer
environments for the maintenance of homeostasis inside body cavities. Differently from

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endoskeletons of our bodies, cuticles have to be exchanged repeatedly during their
growth or metamorphosis, which is one of the common features of ecdysozoans
including nematodes, tardigrades or onychophorans. During the process of molting in
arthropods, mechanical properties of cuticle should be strictly regulated for smooth and
successful completion of whole procedures. The new cuticle which is synthesized
beneath the old cuticle is in many cases soft and colorless. During shedding the old
cuticle, the new cuticle should be soft for shedding of the old cuticle smoothly. The new
cuticle should remain soft for the expansion of the newly formed bodies, but after the
expansion, hardening of the new cuticle begins, and at the same time, cuticle
pigmentation occurs. Generally, cuticles of arthropods are hardened through either
cross-linking reactions (termed tanning or sclerotization), and/or bio-mineralization
process such as calcification.
Calcification of extracellular matrices has been found in multiple types of
organisms like corals, shellfishes, lamp shells or sponges (reviewed in Clark, 2020). As
examples of calcification in arthropods, crustaceans have been well studied and it is
known that calcium carbonate accumulates during cuticle hardening (reviewed in
Nagasawa, 2012; Roer and Dillaman, 1984). There have been also studies on calcium
phosphate accumulation in hard cuticles like mandible in malacostracan crustaceans
(Bentov et al., 2016; Vittori et al., 2018). In crustaceans, both exo- and endo-cuticles are
calcified strongly. The exo-cuticle is also often hardened partially by tanning. On the
other hand, the epicuticle is tanned structure with lipoprotein that may serve as
waterproof barrier (Dennel, 1947a; Roer and Dillaman, 1984; Nagasawa, 2012) (tanning
systems like those observed in insects are discussed in section 4.4). It is also proposed
that disulfides are involved in cuticle formation in decapods (Stevenson, 1969).
Cuticle calcification is observed also in Diplopoda, one of major classes in
Myriapoda (Blower, 1951; Borrell, 2004), but in chilopods (centipedes), another major
class of Myriapoda, cuticles are hardened without calcification. It is thought that the
cuticle of chilopods is hardened/stabilized through the process of “aromatic tanning” for
making crosslinks among cuticular components. The system of the aromatic tanning
seems to be possessed by both chilopods and diplopods (Rajulu and Krishnan, 1968),
and it has been shown that the precursor molecules for cuticle sclerotization have
physicochemical properties different from those used in other arthropods (Blowler,
1950). In aromatic tanning, proteins with a high tyrosine content are probably

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associated with the crosslink formation, most likely via oxidative coupling of tyrosine
residues (Brown, 1950; Blowler, 1951). Crosslink formation of cuticular components
through the chemical modifications of tyrosine residues was shown in a non-arthropod
ecdysozoan, Caenorhabditis elegans (Edens et al., 2001). In the scheme proposed for C.
elegans, tyrosine residues in cuticular proteins are oxidized to phenoxyl radicals by
dual-oxidase (duox), and the resultant phenoxyl radicals couple with other tyrosine
residues to make crosslinked structures such as di-tyrosine or tri-tyrosine. Dual oxidase
is a membrane protein with extracellular peroxidase domain and intracellular NADPH
oxidase domain. It is known that di-tyrosine and tri-tyrosine emit blue fluorescent under
UV radiation, and the blue fluorescent characteristic to di-tyrosine or tri-tyrosine are
found in cuticular structures of crustaceans such as mandibles and/or inter segmental
parts (reviewed by Welinder (1975) or Michels et al. (2016)) as well as in the cuticles of
scorpion and centipede (Govindarajan and Rajulu, 1974; Rajulu, 1971). These studies
suggest the widespread use of di- and tri-tyrosine-mediated crosslinks in these
arthropods.
In chelicerates, it has been reported that hard structures like fangs, chelicerae,
claws or stings contain zinc and manganese ions (Schofield et al., 2003; Gallant et al.,
2016; Politi et al., 2016). In addition, tanning reactions also occur in the cuticle of
chelicerates. The involvement of sulfur containing amino acid residues in strengthening
the cuticle has been considered. In the tick cuticle, the crosslink formation by quinonoid
tanning agents has been suggested for the hardening mechanism of cuticle (reviewed by
Neville (1972) or Hackman and Filshie (1982)). It has been thought that emergence of
fluorescence (emitted by UV radiation) in the cuticle of scorpions is positively
correlated to the increase of cuticle hardeness, which is inferred from the observation
that freshly molted scorpions and the first instar (soft) juveniles do not emit
fluorescence (Pavan and Vachon, 1954; Lawrence, 1954; López-Cabrer et al., 2020). As
substances responsible for the fluorescence, Stachel et al. (1999) identified -carboline
from the cuticle of the scorpions, Centuroides vittatus and Pandinus imperator, and
then Frost et al. (2001) identified hymechrome from the cuticles of multiple scorpion
species.

2.2 Cuticle hardening in insects

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In insects, bio-mineralization with calcium has been observed in 1) puparium cuticles of
dipterans (Fraenkel and Hsiao, 1967; Bodnaryk, 1972), 2) exocuticles of lepidopterans
and hymenopterans (summarized by Hackman (1964)) or 3) rigid areas of the adult
cuticles of coleopterans (Leschen and Cutler, 1994). Calcification seems to contribute to
increase the cuticle strength (Bodnaryk, 1972), though it has been regarded generally
that calcification is very rare in insects (Luquet, 2012). A recent study shows that the
leaf-cutter ant, Acromyrmex echinatior, utilizes accumulation of Mg-calcite (Li et al.,
2020). At the outer surface of cuticles of this ant, Mg-calcite is crystalized, and this
structure really contributes to mechanical protection from enemies or to competition
with other species of ants. The authors of this report also claim that the accumulation of
calcite is much more widely spread in other insects too. Differently from crustaceans, in
insects, high amount of zinc and manganese accumulation is observed at pointed and
hard structures like the tips of mandibles or ovipositors (Fontaine et al., 1991; Quicke et
al., 1998; Schofield, 2001; Schofield et al., 2002), which is like chelicerates. Though
this is not the case with ecdysozoan, the nereid annelid, Nereis virens, has similar
characteristic with the tips of jaws contain high amount of zincs. It has been proposed
that in this annelid, histidine residues on the surfaces of proteins in the jaws are used to
form coordinated structures like Zn[His] 4 and/or Zn[His]3Cl (reviewed in Rubin et al.,
2010). Recently, there have been discussions on involvement of interaction between
zinc and histidine residues in the hardness of the mandibles of not only in annelid, but
also in chelicerates and insects (reviewed in Schofield et al., 2021). The contents of zinc
are too high for each atom to be bound to four histidines in the typical zinc binding
motifs like Zn[His]4 . To overcome this constraint, the authors have proposed a new
binding mode, in which zinc bounds to a single histidine and water as amorphous zinc
hydroxide.
Oxidative polymerization of catecholamines is the major mode of hardening and
stabilization of cuticle in insects (reviewed in Neville, 1972; Hopkins and Kramer,
1992; Sugumaran, 2010; Andersen, 2010; Noh et al., 2016a; Arakane et al., 2017). Fig.
1A and B shows the chemical pathway for cuticle maturation that is divided into two
parts, pigmentation (Fig. 1A) and sclerotization (Fig. 1B). The starting molecule for
both pathways is tyrosine (Fig. 1A). Tyrosine is hydroxylated to dopa by tyrosine
hydroxylase (TH), and dopa is decarboxylated to dopamine (DA) by dopa
decarboxylase (DDC). Dopa and DA are the substrates of oxidation reaction catalyzed

5
by multi-copper oxidase2 (MCO2) for melanin synthesis. For cuticle sclerotization (Fig.
2B), DA is further converted to N-acetyldopamine (NADA) and N--alanyl-dopamine
(NBAD) with the enzyme activities of arylarkylamine N-acetyl transferase (aaNAT) and
N--alanyl-dopamine synthase (NBADS or ebony), respectively. As in melanin
synthesis, NADA and NBAD are used as substrates of oxidation reactions catalyzed by
MCO2. For NBAD synthesis, -alanine is provided by decarboxylation of aspartate that
is catalyzed by aspartate-1-decarboxylase (ADC or black). From NBAD, DA and
-alanine are regenerated by the hydrolysis reaction catalyzed by NBAD hydrolase
(NBADH or Tan). The genes for enzymes involved in these reactions have been
characterized genetically in many insect species. A thorough and comprehensive
analyses of the genes associated with this pathway (TH, DDC, MCO2, aaNAT, ADC
and NBADS) have been performed in two beetles, Tribilium castaneum and Tenebrio
molitor (Arakane et a., 2005, 2009; Gorman and Arakane, 2010; Noh et al., 2015, 2016a,
b; Mun et al., 2020; reviewed in Noh et al., 2016 and Arakane et al., 2017). For both
cuticle pigmentation and sclerotization pathways, oxidations of the catechols (dopa, DA,
NADA and NBAD) are catalyzed by MCO2, as the primary step. Dopa and DA are
oxidized to Dopa quinone and DA quinone, respectively for the melanization pathway,
and NADA and NBAD are oxidized to NADA quinone and NBAD quinone,
respectively for the sclerotization pathway. The same set of oxidation reactions can also
be performed by another type of copper enzyme, phenoloxidase (PO) that has been
characterized extensively for its involvement in innate immune reactions (described in
section 3.2). In melanin synthesis, dopa quinone and DA quinone are non-enzymatically
converted to dopachrome and DA chrome, respectively, and then processed to form
5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI),
respectively. In mammalian system, the conversion of dopachrome to DHICA is
catalyzed by dopachrome tautomerase, which is a simple isomerization reaction. But in
insects, dopachrome is converted to DHI by a decarboxylative rearrangement (Aso et al.,
1989; Sugumaran and Semensi, 1991; Cherqui et al., 1998). The enzyme responsible for
the catalysis of this reaction is termed dopachrome decarboxylase/tautomerase as well
as dopachrome conversion enzyme (DCE). DCEs have been characterized from many
insect systems, and they are homologous to the predicted protein sequence of a yellow
gene (yellow-y) of the fruitfly, Drosophila melanogaster (Johnson et al., 2001), but the
yellow-y gene product itself does not possess any dopachrome

6
decarboxylase/tautomerase activity. The recombinant proteins of yellow-f1 and -f2 of D.
melanogaster on the other hand exhibit the capacity to produce DHI by decarboxylative
rearrangement of dopachrome and also isomerization/tautomerization of DA chrome
and dopa methyl ester chrome (Han et al, 2002). Recent studies indicate the presence of
a separate DA chrome tautomerase activity. The recombinant protein of yellow-h of D.
melanogaster could catalyze the isomerization/tautomerization of DA chrome and
N-methyl-DA chrome, but this enzyme does not act on dopachrome (Barek et al., 2021).
Thus, DHI is the sole product formed from both dopachrome and DA chrome in insects.
It is possible that DHICA can be produced in the presence of divalent metal ions that
would inhibit spontaneous decarboxylation of dopachrome, but this has not been
substantiated in insect so far. In mammalian system, DHI can be produced
spontaneously from dopachrome in certain conditions (summarized in Sugumaran and
Barek, 2018). One of the major differences between mammalian and insect
melanogenesis is choice of the starting substrate. Dopa is used as the starting substrate
by tyrosinase in mammalian system but in insect systems, both dopa and DA (mainly
DA) are used for melanogenesis. Importance of DA in the melanogenesis of cuticle has
been well documented by a number of workers who observed that the spatial patterns of
black pigmentations in cuticle well matched to those of DDC expressions in the
epidermal cells underlying the black part of the cuticles (Futahashi and Fujiwara, 2005;
Ninomiya et al., 2006; Shirataki et al., 2010). In addition, gene knocking down/out
experiments resulted in considerable weakness and delay in melanin synthesis when the
DDC gene was targeted, indicating the important contribution of DA for insect
melanogenesis process (Arakane et al., 2009; Lemonds et al., 2016; Xiao et al., 2020;
Matsuoka and Monteiro, 2018; Zhang et al., 2017). In D. melanogaster, ectopic
expression of TH induced mild melanization that is much enhanced by combining DDC
expression, indicating that DA is preferentially used for melanin synthesis compared to
dopa (True et al., 1999). The preference of DA is also supported by kinetic analyses of
laccase-type enzyme in insect cuticle and endogenous/recombinant MCO2s showing
that DA is better substrates than dopa, based on the parameters including Kcat values
(Arakane et al., 2016; Dittmer et al., 2009; Gorman et al., 2012; Thomas et al., 1989).
In contrast to melanogenesis where unacylated dopa and DA are used to produce
the dark colored melanin pigment, the sclerotization pathway uses only the acylated
derivatives such as NADA and NBAD for hardening/stabilizing the cuticles. Through

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oxidative polymerization of cuticular components, these substances mediate covalent
crosslinks between cuticular proteins and/or between cuticular protein and chitin fibers.
It has been well established that both the aromatic rings and the side chains of
catecholamine derivatives are used for crosslink formation with the surrounding
cuticular components and/or polymerization of the oxidized substrates themselves
(Andersen 2010; Barek et al., 2017; Sugumaran 2010). In the pathway of sclerotization
(Fig. 1B), after oxidation of NADA and NBAD, the ring structures of the products,
NADA quinone and NBAD quinone, respectively, are used for making primarily
quinone crosslinks, but these quinones can also be isomerized through enzymatic or
non-enzymatic processes to become NADA quinone methide and NBAD quinone
methide, respectively. The -carbon atoms of these quinone methides are used to make
side chains’ crosslinks (-sclerotization). NADA quinone methide and NBAD quinone
methide are also further converted by quinone methide tautomerase to dehydro-NADA
and dehydro-NBAD, respectively, that are oxidized by MCO2 or other enzymes to the
corresponding quinones, and the resulting products (,-dehydro-NADA-para-quinone
methide and ,-dehydro-NBAD-para-quinone methide) are used for making
crosslinks with both - and -carbons (,-sclerotization) (Andersen 2010; Barek et al.,
2017; Sugumaran 2010). in vitro experiments confirm the participation of histidine
residues (and other nucleophilic residues) on protein surface in covalent linking with
quinones or quinone methides (Suderman et al., 2006; Mun et al., 2015).
During cuticle sclerotization, dehydration of cuticle can also occur, during which
process, polymers produced by oxidation of NADA and NBAD may fill in the space. It
is widely accepted that NADA does not produce pigmented cuticle in contrast to NBAD
that produces brownish colored cuticle, suggesting the occurrence of different
downstream reactions in each of the pathways with NADA and NBAD (reviewed in
Andersen, 2010, Sugumaran, 2010). Moreover, the ratios of NADA and NBAD may
also influence the chemical structures inside cuticle matrices and thereby affecting the
mechanical properties of cuticle. The data from the phenotypic analysis of T. molitor
indicate the difference in localizations of these two substrates, as has been observed in
the color patterns of the cross sections of elytra taken from the knockdown individuals
(Mun et al., 2020). In knockdown of Tm-aaNAT, blackening was seen at the boundary
between exocuticle and endocuticle (mesocuticle) and slightly at endocuticle, but in
Tm-ebony knockdown, blackening was seen in exocuticle, indicating that NADA and

8
NBAD have their specific roles in mesocuticle (also in endocuticle) and exocuticle,
respectively.
Oxidation of catechols in cuticle can also be achieved by peroxidases, in addition
to MCO2-mediated (or PO-mediated) reactions (Andersen 2010; Barrett 1991; Coles
1966; Hurst 1945; Ito et al., 2020; Locke 1969; Neville 1972). Peroxidases use
hydrogen peroxide as an electron acceptor to oxidize a variety of organic substrates
such as phenols, aromatic phenols, phenolic acids (Buchert et al., 2010). In in vitro
experiments, it was shown that polymerization of model proteins occurred in the
presence of hydrogen peroxide, peroxidase and catechols (Hasson and Sugumaran,
1987). In the experiment with the cuticular protein (MsCP36) from the tobacco
hornworm, Manduca sexta, Suderman et al. (2010) witnessed the protein
polymerization through dityrosine formation catalyzed by peroxidase. The in vivo
analyses have also supported this hypothesis of the peroxidase-mediated cuticle
hardening/stabilization. In insects, like the cases in nematode, it is possible that duox is
responsible for forming crosslinks between cuticular components for stabilization of
cuticle matrices through dityrosine formation. Knockdown of duox in wing discs of D.
melanogaster induced wing decay or collapse over time, supporting this hypothesis
together with the quantification data that dityrosine contents declined in the
duox-knocked down individuals (Anh et al., 2011). ROS production in wings after
eclosion was widely spread in the wings, indicating that dityrosine formation occurs
throughout the wings. It was shown that a neomorphic mutation of duox was
responsible for curly wings in Curly mutant (Hurd et al., 2015), indicating the important
roles of duox in cuticle formation. Other peroxidase genes like Ampxd of the honeybee,
Apis mellifera, or curly suppressor (Cysu) of D. melanogaster, have been invoked for
their possible involvement in cuticle formation process, based on the expression
patterns or the phenotypes of wing malformations (Bailey et al., 2017; Soares et al.,
2011). In the anatomical study, blue fluorescence (an indicator for the presence of di-
and tri-tyrosines) are found in cuticle structures of insects, such as hard parts like
mandibles, indicating presence of cross-link formation via di- and tri-tyrosine formation
(Büsse and Gorb, 2018).
Transglutaminase (TG) mediated crosslink is yet another way to crosslink
proteins. This enzyme catalyzes the formation of a transpeptide bond between residues
of glutamate and lysine. The TG-mediated crosslink structure is found in elastic tissues

9
like lungs or skins of human, in which elastin and fibrillin molecules are polymerized
through the TG-mediated reaction (reviewed in Ozsvar et al., 2021 or Savoca et al.,
2018). In horseshoe crabs, TG-mediated crosslink formation is used in the process of
blood coagulation (Iwanaga, 2002). It was shown in in vitro experiment that proteins
extracted from the cuticles of the horseshoe crab, Tachyples tridentatus, were
crosslinked by TG, suggesting that also in vivo TG could be involved in stabilization of
cuticular matrices by catalyzing polymerization of cuticular components (Iijima et al.,
2005). In insects, it is possible that TG has similar roles, which is assumed from the
knockdown experiment of the gene for TG resulting cuticle malformation in adults of D.
melanogaster (Shibata et al., 2010).
In several species of chelicerates and crusaceans, involvement of sulfur amino
acids in cuticle hardening has been discussed (reviewed in Neville, 1972), but in insects,
it is known that cuticle proteins with sulfur amino acids are rare, though recent
molecular biological studies have revealed that a few families of cuticle proteins
containing cysteine or methionine residues (Willis, 2010). They include CPAP1,
CPAP3 (cuticular peritrophin-A like protein) or CPCFC (cuticular proteins with 2 or 3
C-x(5)-C motifs). CPAP1 and CPAP3 have chitin binding motif consist of six cysteines
that may be linked by disulfide bonds. CPCFCs have repeated sequences of 16 residues
with two cysteines in the motif of C-x(5)-C (Jasrapuria et al., 2010; Willis et al., 2012).
Lepidopteran and Dipteran insects have proteins with YxGGFGGPPG(V/L)L motifs,
and lepidopteran proteins have many C-x(4)-C motifs (Asano et al., 2013). The cysteine
residues seem to form intra motif disulfide bonds, because mobility of this protein in
SDS-PAGE in reducing condition is slightly lower than that in non-reducing condition
(unpublished observation).
It is also important to mention about the precursor of one of the melanin
producing enzymes, prophenoloxidase (proPO). In the silkworm, Bombyx mori, proPO
is transported from the hemolymph to the cuticle after the synthesis in a type of
hemocytes, oenocytoids (Asano and Ashida, 2001a; Iwama and ashida, 1984). During
transportation or after transportation to the cuticle, the methionine residues on the
surface of proPO molecules are oxidized to methionine sulfoxide or methionine sulfone
(Asano and Ashida, 2001b). The reason for such modification is not clear at this point.
Whether such methionine modification has any biological significance or not needs to
be resolved by further studies. The reason why cuticular proteins with sulfur amino

10
acids are rare can be explained by some toxicities or undesired effects of the sulfur
residues, but formation of disulfide bonds or chemical modifications may mask such
bad effects.

3. Phenol-oxidizing enzymes in insect cuticle

In early periods of the studies on biochemical reactions occurring in hardening process
of insect cuticles, diphenols had already been extracted from insect cuticles. Their
correlation with cuticle hardening had not been understood until the first establishment
of the model for sclerotization that called for the oxidation products of o-diphenols as
responsible for hardening of cockroach oothecal and similar reactions occurred in
integuments of insects (Pryor, 1940a. b). Extensive studies carried out later established
that two catecholamine derivatives, NADA (Karlson and Sekeris, 1962) and NBAD
(Hopkins et al., 1982) play a pivotal role in cuticle hardening (the history of the findings
of these sclerotizing agents is summarized by Neville (1972), Andersen (1990), Hopkins
and Kramer (1992) or Sugumaran (1998)). Until 90s, there had been several candidates
of enzymes suggested to participate in cuticular sclerotization by oxidizing these
catecholamine derivatives. These include, laccase-type phenoloxidases, tyrosinase-type
phenoloxidases, granular phenoloxidase or other enzymes with oxidase activity against
diphenols (summarized by Andersen (1990), Ashida and Yamazaki (1990) or Barrett
(1991)). Among the candidates, the former two enzymes have been well-characterized
in the recent two decades, along with the development of the techniques in molecular
biology or molecular genetics. The rest of this section reviews the details of the studies
on the two enzymes, currently known as MCO2 (laccase-type) and PO
(tyrosinase-type).

3.1 Laccase-type enzymes

3.1.1 Overview of laccases and multi copper oxidases

Laccase was originally found in the Japanese lacquer tree, Rhus vemicifera
(Yoshida,1883; reviewed in Sumner, 1947). The name, “laccase” was first used by
Bertrand who studied laccase from the Indo-China lacquer tree, Rhus succedanea
(1894a, b). Then, Bertrand (1895) and Laborde (1896) found laccase activity in multiple
plants and fungi. Since then, laccases have been shown to be widely distributed in

11
plants and fungi. Laccases are one of the oldest enzymes ever described (Zumarraga et
al., 2007; Kunamneni et al., 2008; Rodriguez-Couto, 2012; Shiba, 2002). Extensive
studies carried out on laccases resulted in the accumulation of a large amount of
knowledge on their molecular properties/structures, mechanisms of catalysis, functions
of laccase genes and molecular evolution. Laccases have received lot of attention also
due to their potential use for practical purposes. The important characteristics of
laccases are 1) their capacity to oxidize variety of substrates like o- and p-diphenols,
polyphenols, aminophenols, aromatic amines and aliphatic amines, and 2) their high
stabilities in harsh conditions such as extreme pHs or high temperature (Buchert et al.,
2010; Janusz et al., 2013; Nakamura and go, 2005; Sharma et al., 2007). Laccase is
classified to three-domain multi-copper oxidases (3dMCOs) that have three consecutive
cupredoxin domains (Fig. 2). There are variety of proteins composed of cupredoxin
domains, and they form the protein superfamily of MCO. In the MCO superfamily,
3dMCOs form the most diverse group of enzymes. The group of 3dMCOs includes
laccase, white laccase, ascorbate oxidase, ferroxidase, and bacterial proteins like
manganese oxidase. It has been demonstrated that 3dMCOs have variety of biological
functions including the biosynthesis of lignin polymers from lignin monomers,
degradation of lignin polymer, pigment production, fruiting body formation,
detoxification and stress defense (Nakamura and go, 2005; Janusz et al., 2020; Sharma
et al., 2007). The origin of 3dMCOs can go back to prokaryotes, and the diverse
functions of 3dMCOs found in each of animals, plants or fungi might have evolved
independently in each of taxa. In molecular phylogenetic trees, animal 3dMCOs are
nested within the cluster of fungal laccases that is isolated from the cluster of plants’
3dMCOs (Hoegger et al., 2006; Janusz et al., 2020; Nakamura and Go, 2005; Sharma et
al., 2007). The laccase-like properties of the enzymes found in insect cuticle might have
evolved independently from evolution of laccases in the linage of plants, and the insect
laccases do not seem to be orthologous to plant laccases.

3.1.2 Laccase-type enzymes in insect cuticles

In the early studies on enzymes involved in cuticle hardening of insects, there had been
a hypothesis that p-quinones might mediate formation of crosslinks among cuticular
components, resulting in hardening of cuticles (summarized by Neville (1972)). Though
involvement of p-diphenol in cuticular hardening has not yet been established, cuticular

12
enzymes capable of p-diphenol oxidation have been well characterized. The
phenol-oxidizing enzyme in insect cuticle with activity against p-diphenols was first
described by Onishi (1954) (reviewed in Andersen, 1990 or Ashida and yamazaki,
1990), who noticed the presence of enzyme activity to oxidize
dimethyl-p-phenylendiamine in the white puparia of the fruit fly, Drosophila virilis.
The activity was further characterized by Yamazaki (1969), and the enzyme was
identified to be a laccase-type protein by the enzymological definition of Keilin and
Mann (1939) and Dawson and Tarpley (1951). The characteristics of the laccase-type
enzyme are 1) the ability to oxidize p-phenylendiamine and polyphenols but not
tyrosine or p-cresol, 2) the insensitiveness to carbon monoxide and 3) the inhibition by
potassium cyanide and sodium diethyldithiocarbamate. In B. mori and D. virilis,
laccase-type activity appears in the very time when sclerotization occurs in pupal and
puparium cuticles, respectively (Yamazaki, 1969, 1972). Since these initial findings,
several studies have described the characterization of laccase-type activities in insect
cuticles including the blowflies, Calliphora vicina (Barrett and Andersen, 1981),
Lucilia cuprina (Barrett, 1987a), the flesh fly Sarcophaga bullata, the blood sucking
bug, Rhodnius prolixus and T. molitor (Barrett, 1987b), the desert locust, Schistocerca
gregaria (Andersen, 1978), M. sexta (Thormas et al., 1989) and D. melanogaster
(Sugumaran et al., 1992) (reviewed in Andersen (1990), Ashida and Yamazaki (1990)
or Barrett (1991)). Dittmer and Kanost (2010) summarized kinetic parameters of the
laccase-type enzymes in insect cuticles and compared them to those obtained in the
recent analyses of the purified and recombinant MCO2s. One of important
characteristics found in the laccase-like enzymes is the presence of systems for temporal
regulation of the enzyme activity during molting or puparium formation. In D. virilis, it
was shown that the activity of the laccase-type cuticular enzyme appeared just before
puparium formation and it reached maximum at 3 hours after puparium formation. In B.
mori, it was shown by measurement with manometer to quantify oxygen consumption
that laccase activity began to appear after pupal ecdysis. These observations had led to a
hypothesis that the laccase-type enzymes accumulated as inactive forms and were
activated to initiate hardening of puparium or pupal cuticles (Yamazaki, 1969, 1972).
Since laccase-type enzymes in insect cuticles bind tightly to cuticular matrices, it
required the use of solubilizing agents and long period of extractions to collect their
enzyme activity in soluble fractions (Andersen, 1990; Ashida and Yamazaki, 1990). In

13
the study of B. mori (Yamazaki, 1989), -chymotrypsin was used for the extraction of
the precursor of laccase-type enzyme from the newly ecdysed pupal cuticles. The
purified precursor protein from the extract showed little sign of enzyme activity after
gel electrophoresis when stained with dimethyl-p-phenylenediamine. But the
proteolytically treated enzyme readily exhibited laccase type staining on gels. This
suggests that the enzyme is present as an inactive precursor in vivo and that proteolytic
processing results in the activation of the precursor. It should be noted here that
originally Yamazaki (1972) purified and characterized the laccase-type enzyme only
after solubilizing it with trypsin from the newly ecdysed pupae of B. mori. The purified
enzyme showed apparent enzyme activity, which is explainable by activation of the
precursor during the solubilization process using trypsin. Like B. mori, in the study of
laccase-type enzyme from the same lepidopteran insects, M. sexta, Thomas et al. (1989)
also used trypsin treatment of pharate pupal cuticles to extract the laccase-type enzyme.
After partially purifying the enzyme, they determined several of its properties. The
Manduca laccase-type enzyme preparations showed not only oxidization activity
against catechols, but also exhibited the ability to produce -hydroxylated products
(-hydroxylation of catecholamines is described in later section). Until the early 90s,
several authors have reported the partial purification and characterization of
laccase-type enzymes, but for almost 12 years, and determination of their amino acid
sequences, cDNA cloning and identification of genes encoding these proteins had
remained unexamined. In 2000s, the first report of cDNA cloning of 3dMCOs from
insects appeared in the biochemical literature (Dittmer et al., 2004). Since then, the
studies on MCO2-mediated cuticle formation became active, and finally two groups
reported identification of genes for the laccase-type enzymes that had been
characterized in B. mori (Yamazaki, 1972, 1989) and M. sexta (Thomas et al., 1989).

3.1.3 Biochemical analyses of MCO2s

Dittmer et al. first reported the cDNA cloning of the genes for insect 3dMCOs in 2004.
By using degenerate primers designed for the conserved sequence motifs of plant
laccases and related proteins, cDNA fragments were amplified and then the sequences
of the full-length cDNA of the first of 3dMCOs in metazoans was obtained. These
authors identified two types of protein sequences, MCO1 and MCO2. From A. gambiae,
AgMCO1 was identified, and from M. sexta, MsMCO1 and MsMCO2 were identified.

14
The structures of these proteins were different from 3dMCOs that had been previously
described in other organisms by the presence of N-terminal extensions followed by the
C-terminally located catalytic domains (Fig. 2A). In the N-terminal extensions of all the
three proteins, cysteine-rich sequences are present, and AgMCO1 has von-willebrand
factor sequences that are postulated to be involved in protein-protein interaction (Bork,
1993) or immune reactions of insect hemocytes (Goto et al., 2001; Kotani et al., 1995).
Among the three genes, MsMCO2 was identified as the candidate gene involved in
cuticle hardening, judging from its expression in epithelial tissues during pupal molt.
The expression of MsMCO2 is very strong in the stage of pharate pupa, but in day 0
pupa, the expression is very weak. In several reports published later on, the same pattern
- strong expression in pre-ecdysal stages and decline of expression after ecdysis - has
been observed (Futahashi et al., 2010, 2011; Masuoka et al., 2015 or Mun et al., 2020;
Yatsu and Asano, 2009). The strong expression in pre-ecdysal stages indicates that
MCO2 accumulates mainly in the exocuticle that is secreted before ecdysis. This is
consistent with observations that the outer part of the cuticle, the part produced earlier
than inner part, is heavy pigmented/sclerotized (Masuoka et al, 2013; Mun et al., 2020;
reviewed by Neville (1975) or Andersen (2010)). Later, in double-stranded
RNA-mediated knockdown experiments, Arakane et al. (2005) have shown that the
gene for MCO2 (TcMCO2) is involved in both cuticle pigmentation and formation in
Tribolium. The phenotypes are defects in cuticular pigmentation and malformation, the
latter of which is probably caused by incomplete cuticle hardening.
Following these studies, two reports appeared on amino acid sequence analyses of
the laccase-like enzymes in the cuticles of two lepidopteran insects, B. mori and M.
sexta. In Bombyx study (Yatsu and Asano, 2009), trypsin-solubilized enzyme was
purified to near homogeneity as evidenced by the presence of a single band of 70k, on
SDS-PAGE electrophoretogram in contrast to the previous purification that contained
three bands (70k, 66k and 62k) (Yamazaki, 1972). Using the Edman degradation and
LC/MS analyses of the purified protein, information of the internal amino acid
sequences was obtained, and cDNA for the purified protein was identified (Yatsu and
Asano, 2009). The predicted protein from the cDNA (BmMCO2) has high sequence
identity (95%) with MsMCO2 (Dittmer and Kanost, 2010). The N-terminal sequence
(NPALS) corresponds to Arg147-Ser151 of the predicted sequence, indicating that the
N-terminal 146 amino acids are depleted in the purified protein, suggesting that the

15
N-terminal part from the Arg146 was lost during extraction by trypsin-treatment.
Similar to MsMCO2, the expression of BmMCO2 is strong before pupal ecdysis,
indicating that BmMCO2 protein accumulates in the newly synthesized cuticle before
pupal molt. The enzyme activity corresponding to laccase-type was assayed by in situ
staining of the pupal cuticles using DA as the substrate. In this analysis, the activity of
another phenol-oxidizing enzyme, PO, was inactivated by heat treatment, which utilized
the character of laccase-type proteins in insect cuticles that they are highly resistant
towards heat treatment (reviewed in Ashida and Yamazaki, 1989 and Barrett, 1991).
The pupal cuticles taken from prepupae and just-ecdysed pupae were not stained with
DA, but those from later stages were stained to black, indicating that laccase-type
activity in the cuticles is very low in earlier stages, but after molting the activity is
highly elevated (Yatsu and Asano, 2009). It is thought that MCO2 accumulating in the
new cuticle is present as an inactive precursor and is activated after pupal ecdysis.
However, another possibility, that there are some factors which inhibit MCO2 activity
until the time when MCO2s are supposed to start cuticle pigmentation/sclerotization
cannot be ruled out at this time (Dittmer et al., 2009; Dittmer and Kanost, 2010; Yatsu
and Asano, 2009).
To re-examine the characteristics of the precursor protein of laccase-type enzyme
from B. mori our group conducted a detailed analysis (Asano et al., 2014). First, we
solubilized the laccase-type enzyme with -chymotrypsin treatment according to
Yamazaki’s (1989) procedure. Then we analyzed the enzyme for its biochemical
properties. The purified BmMCO2 precursor has the mobility of 81k in SDS-PAGE
(Fig. 3). The N-terminal sequence (RNPALS) corresponds to Arg146-Ser151 of the
predicted sequence, indicating that the N-terminal 145 amino acids are depleted in the
purified protein (Fig. 4A). It is possible that the N-terminal part was lost during its
extraction with -chymotrypsin. In the enzyme assay, the BmMCO2 precursor was not
inactive completely, but after treatment with trypsin the precursor became a more active
form showing 17-fold change. By this treatment, the mobility in SDS-PAGE shifted
from 81k to 70k (Fig. 3, left panel). Also, in native-PAGE analyses (Fig. 3, middle
panel), clear signal of enzymatic activity can be seen in the sample pre-treated with
trypsin. In Edman degradation analysis, the cleavage site by trypsin treatment was
determined to be the peptide bonds at C-terminal region (Fig. 4A), but it has not been
shown that proteolytic processing really occurs in vivo. When BmMCO2 precursor was

16
incubated in the buffer containing iso-propanol, the precursor was activated as can be
seen in the activity-staining gel (left lane of Fig. 3, right panel), and the activity after
this treatment is comparable (0.6-fold) to that of the BmMCO2 precursor treated with
trypsin (Fig. 4B). By the interaction with iso-propanol, conformational changes might
have been induced in the precursor without proteolytic processing.
After the report for the identification of the gene for laccase-type enzyme in B.
mori (2009), there was the report of the amino acid sequence analysis of MCO2 that
was purified from the pharate pupal cuticles of M. sexta (Dittmer et al., 2009). As
described, in Manduca, it had been shown that cuticles of pharate pupae contained
laccase-type enzyme (Thomas et al., 1989), and its enzymological analyses were
performed with the partially purified enzyme that was obtained from the cuticle extract
prepared by trypsin digestion. In the report of 2009, cuticle extract was prepared by the
treatment with -chymotrypsin. The purified protein (referred to endogenous
MsMCO2) had the N-terminal sequence of RRNPALSAPD (Arg119 – Asp128), almost
the same sequence with those determined in the trypsin-solubilized BmMCO2 and
BmMCO2 precursor. Like the cases in BmMCO2, it is thought that the N-terminal part
was lost in the endogenous MsMCO2. By baculovirus system, the authors synthesized
recombinant proteins of full length of MsMCO2 (FLrMsMCO2) and that lacking the
N-terminal 106 amino acids (106rMsMCO2). In western analysis of the MsMCO2s
and cuticular samples with the specific antibody, the mobility of FLrMsMCO2
corresponded to that detected in samples prepared from pupal cuticles, indicating that
MsMCO2 is present in unprocessed form. The mobility of the endogenous MsMCO2 is
almost the same with that of 106rMsMCO2, indicating that -chymotrypsin might
have cleaved the peptide bond at 118-119 during the process of its solubilization. The
three forms of MsMCO2s (endogenous, FLrMsMCO2 and 106rMsMCO2) show
enzyme activities without any treatments for activation, which is consistent with the
observation that the purified BmMCO2 precursor is enzymatically active. The specific
activities of FLrMsMCO2 and 106rMsMCO2 are not largely different, indicating that
the presence or absence of the N-terminal extension does not affect the strengths of
MsMCO2 activity. When compared with recombinant MsMCO2s, the endogenous
MsMCO2 showed higher activity than the recombinant proteins (the graph in the right
panel of Fig. 4B was drawn according to the graph in the reference (Dittmer et al.,
2009)). From this graph, it can be speculated that a portion of endogenous MsMCO2 is

17
activated. In M. sexta, pigmentation of pupal cuticle starts in pharate pupal stage, and
the endogenous MsMCO2 was purified from cuticles of pharate pupae. In the analyses
of BmMCO2, the precursor was activated by artificial treatments, but currently
endogenous factors possibly involved in MCO2 activation in vivo are being
characterized (Asano and Kanost, in preparation).
By using large lepidopteran insects, biochemical characteristics of the MCO2
proteins were analyzed by the two groups, but Kanost’s group has also characterized the
MCO2s from other insect orders using recombinant proteins (Lang et al., 2012; Gorman
et al., 2012). It had been shown that in T. castaneum TcMCO2 has two alternatively
spliced isoforms, TcMCO2A (A-type) and TcMCO2B (B-type) (Arakane et al., 2005).
As shown with the genomic structure of BmMCO2 (Fig. 2B), in many insect species,
genes for MCO2s are expressed at least in two splicing isoforms. The N-terminal part
including over half of the catalytic region is common among the isoforms, but the
remaining C-terminal parts are encoded in different exons and the amino acid sequences
of the C-terminal regions are different. It was shown that in T. castaneum the expression
patterns for the two isoforms were different, and each of the isoform specific
knockdowns resulted in lethal phenotype, indicating that each of the two isoforms has
its own important roles. By using recombinant proteins, Lang et al. (2012) characterized
an isoform (A-type) of MCO2 from D. melanogaster and Gorman et al. (2012)
characterized A- and B-isoforms from both T. castaneum and A. gambiae. Like
MsMCO2s and the purified BmMCO2 precursor, all the recombinant proteins
(DmMCO2A, TcMCO2A, TcMCO2B, AgMCO2A and AgMCO2B) are enzymatically
active. The recombinant TcMCO2A and B showed different enzymological properties,
and the most notable difference is that TcMCO2A had higher enzyme activity. Between
the two MCO2 isoforms of A. gambiae (AgMCO2A and B), pH preference is the
notable difference, also suggesting the functional diversification of MCO2 in this
species. In D. melanogaster, there are over three-types of splicing isoforms (Flybase:
https://flybase.org), indicating diversification of molecular functions and more
complexed regulation of the gene expression.

3.1.4 Phenotypic analyses of the genes for MCO2s

Nowadays, molecular biological techniques are very popular, and knockdown
experiments or genome editing have become to be widely utilized in insect research. In

18
2004, the cDNA cloning of MCOs from insects were firstly reported in the analyses of
M. sexta and A. gambiae, and next year there was a report on knockdown analysis of
genes for phenol-oxidizing enzymes in T. castaneum. Following this work, a number of
groups reported the phenotypic analyses of MCO2 knockdown. The species used were
the pine sawyer beetle, Monochamus alternatus (Niu et al., 2008), A. mellifera
(Elias-Neto et al., 2010), the stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius
(Lygaeodae), and Megacopta punctatissima (Plataspidae) (Futahashi et al., 2011).
Futahashi et al. (2010) also reported a correlation between the patterns of body colors
and MCO2 expression in the swallowtail butterfly, Papilio Xuthus. After these
pioneering reports, many studies on the gene functions of MCO2s have been published
(summarized in Table. I). In most cases, the knockdown of MCO2 led to malformed
cuticle and defects in melanotic cuticle pigmentation. Coleopteran insects are well
known for their high efficiency of double-stranded RNA mediated knockdown and this
methodology has been applied to many species in this order to show the functions of
MCO2 genes. With the clear visible phenotypes in body color pigmentation, MCO2 was
selected as one of markers to see the effects by genome editing (Watanabe et al., 2012).
Microinjection of mRNAs for zinc-finger nuclease or transcription-activator-like
effector nuclease into the cricket, Gryllus bimaculatus, resulted in efficiencies of 48%
and 17%, respectively, in generations of founder animals with alleles of the target gene
disruptions.
In A. gambiae, expression of an isoform of MCO2 (AgMCO2A) is induced after
inoculation of bacteria (Gorman et al., 2008), and in Anopheles sinensis, survivability
after injection of bacteria was decreased by knockdown of MCO2 gene (AsMCO2) (Du
et al., 2017). The knockdown of MCO2 gene in T. castaneum resulted in lowered
resistance against infection by pathogens (Hayakawa et al., 2018). These results are
consistent with the previous observation that an ebony mutant of Drosophila (the
amount of the sclerotizing agent, NBAD, is insufficient) is less resistant against
infection (Flyg and Boman, 1988; Lemaitle et al., 1996). Weakened cuticle strength
might be a cause of the higher susceptibility to infection, or as speculated in the study of
A. sinensis (Du et al., 2017), MCO2 would be involved in melanin production as
defense reactions against pathogens.

3.1.5 Programs for cuticle formation

19
Biochemical analyses and direct measurements/detection of laccase-type activities in
cuticles have indicated the presence of systems for regulation of MCO2 activity (Asano
et al., 2014; Yamazaki. 1969, 1972, 1989; Yatsu and Asano, 2009). These experiments
focused on pupae and puparia, and cuticle hardening of these cases seems to proceed
uniformly in whole body, while as can be exemplified by eclosion of hemi-metabolous
insects (Fig. 5), timing of cuticle hardening seems to be different in each body part.
From the last instar of nymphs of the cicada (Fig. 5A), adult is emerging as in photo-1.
After the adult sheds all the legs from the old cuticles, it keeps the posture for several 10
minutes (photos-2), which seems to be a period during the legs being hardened enough
to support its body in the next step (photos-3). Then, the adult holds the nymphal exuvia
with the newly formed legs and start to expand the wings (photos-4). During the
expansion, the wings seem to keep the softness, but after the wings fully expanded,
cuticle hardening (and pigmentation) seems to start in the cuticles of whole body
(photos-5). In the measurements with the device to quantify vending forces, it was
observed that hardening of legs started first, and then that of wings and other body parts
started after wing expansion (unpublished data) (this ordered cuticle hardening is
illustrated in Fig. 5B). In addition, during the period of leg hardening (photo-2), the
adult did not move, and only after the legs hardened enough, the adult hold the exuvia
and then expanded the wings, indicating that both the program of behaviors and that of
metabolic pathways for cuticle hardening are required to be orchestrated for proper
formation of the adult cicada body, though it is not known what kinds of mechanisms
are involved in the regulation of this orchestration.
In D. melanogaster, the very recent study indicates that insulin-like peptide 8 of
Drosophila (dilp8) and its receptor, leucine-rich repeat containing G-coupled receptor 3
(lgr3), are involved in strict control of morphogenesis by regulating behavioral
programs and possibly metabolic programs for cuticle sclerotization. During white
pupal formation, contraction of the larval muscle is necessary for puparium
morphogenesis in which the length in anterior-posterior direction is shortened. This
process is governed by neural cells in central nervous system expressing lgr3 after
reception of dilp8 that is secreted by epithelial cells in response to increase of ecdysone
titer (Heredia et al., 2021). During the hardening of puparium cuticles, the muscles
continue contraction status probably to keep the shortened shape until the puparium
cuticle to be hardened. The authors think that the metabolic pathway for cuticle

20
hardening might be suppressed at the early phases of puparium formation by the
dilp8-lgr3 pathway to maintain the larval cuticle soft enough to be shortened. Like
muscle activity or glue spreading that are controlled strictly by dilp8-lgr3-dependent
neural activity (Heredia et al., 2021), metabolisms for cuticle hardening like synthesis of
MCO2 substrates or activation of MCO2 might be controlled to start in proper timings
in consonance with the behavioral programs. There is another recent study related to
this point. Bursicon is known as a peptide hormone that regulates cuticle
tanning/pigmentation, but this factor is also involved in behaviors like wing expansion
after eclosion (Luo et al., 2005). Like dilp8-lgr3 pathway, bursicon seems to act to
neuronal cells in ventral nerve cord to initiate cuticle maturation (sclerotization and
pigmentation), which is different from the past view that bursicon directly acts to
epidermal cells to initiate cuticle maturation (Flaven-Pouchon et al., 2020).

3.2 Tyrosinase-type enzyme

3.2.1 Overview of tyrosinase-type enzyme in insects

The tyrosinase-type enzyme of insects is currently called as “phenoloxidase (PO)”. POs
have enzyme activities of both monophenol monooxygenase and catechol oxidase,
which is the characteristics of tyrosinases from other organisms. It is thought that
melanization reactions mediated by PO is one of the immune reactions in arthropods
like insects and crustaceans. Dopa and DA are oxidized to the corresponding quinones
that are highly reactive and possibly toxic to microorganisms or parasites invading into
the hemocoels or cuticular matrices of host insects. In normal physiological conditions,
PO is present as inactive precursor, prophenoloxidase (proPO) in both hemolymph and
cuticle, and is activated by limited proteolysis through the action of serine protease
cascade that is triggered by microbial cell wall components like peptidoglycan or
-1,3-glucan. These pathogen-derived substances are recognized by their specific
recognition proteins like peptidoglycan recognition protein (PGRP), -1,3-glucan
recognition protein (GRP) or gram-negative binding protein that activate the cascade
reactions. After triggered by recognition of pathogens, the zymogens of cascade
components (serine proteases) are sequentially activated, and finally proPO is
proteolysed at the peptide bond at C-terminal side of arginine in NR-FG like motifs that
locate at around 50th residues from the N-terminus (Kawabata et al., 1995). It is

21
important that the serine protease cascades activate not only proPO, but also toll
signaling pathway for synthesis of anti-microbial peptides. In B. mori, it was shown that
a component of the serine protease cascade (BAEEase) is orthologous to späztle
processing enzyme of D. melanogaster that cleaves pro-form of the toll ligand, späztle
(spz), to produce its mature form (Yoshida and Ashida, 1986; Jang et al., 2006). In M.
sexta, hemolymph protease 6 activates proenzymes of two proteases, hemolymph
protease 8 and proPO activating protease 1 (PAP1), that are involved in Toll activation
and proPO activation, respectively (Chunju et al., 2009) (the details of serine protease
cascade for immune reaction in arthropods are reviewed by Cerenius et al. (2010),
Kanost et al. (2004), Kanost and Gorman (2008), Iwanaga and Lee (2004) or Ryu et al.
(2010)). Identification of the genes/factors in the protease cascades have been done
mainly through the biochemical studies of hemolymph samples from multiple species or
through Drosophila genetics. Since the early periods of studies including the
purification of proPO from the hemolymph of B. mori (Ashida, 1971), there had been
many studies on purification and molecular characterization of proPOs (summarized in
Ashida and Yamazaki, 1990; Ashida and Brey, 1998). In 1995, three reports
simultaneously first outlined the cDNA cloning of insect proPOs from B. mori
(Kawabata et al.199 5), M. sexta (Hall et al. 1995) and D. melanogaster (Fujimoto et
al.1995) following the characterization and cDNA cloning of a crustacean proPO
(Aspán et al., 1995). These studies established that proPOs are homologous to arthropod
hemocyanin (hemocyanin: Hc). By gel permeation column chromatography and
high-performance liquid chromatography, the proPOs from B. mori and M. sexta were
shown to be heterodimers and Drosophila proPO A1 was shown to be a homodimer
(Fujimoto et al., 1993; Jiang et al., 1997; Kawabata et al., 1995). But the subunit
compositions appear to vary, for instance, depending on ionic strength and other factors.
Thus, for example the proPOs from the cecropia moth, Hyalophora cecropia, and the
flesh fly, S. bulluta, appear to be monomeric in nature (Andersson et al, 1989; Chase et
al., 2000). Crystal structure of Manduca proPO was solved (Li et al., 2009) and it was
simulated in silico that the cleavage of NR-FG site by the activating proteases showed
drastic switch of the electronical charges around the cleavage site from negative to
positive, which might be important for interactions with other immune factors. When
the cleavage site for proPO activation was determined in proPO of B. mori (Kawabata et
al., 1995), the authors used the activating protease (proPO activating enzyme: PPAE)

22
that had been purified from the larval cuticles. Probably in B. mori, because of the ease
in purification from cuticle samples, PPAE from the cuticle had been well characterized
(Ashida et al., 1974; Dohke 1973a, b; Satoh et al., 1998). Cuticular factors for proPO
activation including PAP1 were studied also in M. sexta (Aso et al., 1984; Gupta et al.,
2005).

3.2.2 Molecular identification of tyrosinase-type enzyme in insect cuticle

At wounded parts of the cuticle, melanin is produced probably to cover wounded parts
to prevent loss of water and invasions by pathogens. As early as 1870, Pasture described
the presence of melanized wound marks on the cuticular surface of Bombyx larvae,
which were caused by crowd rearing. The wound marks are inflicted by silkworm
themselves when they climb over each other (Pasteur, 1870; reviewed in Ashida and
Brey, 1997). As a pioneering biochemical study, Lai-Fook proposed the detailed
scheme for cuticle hardening and melanin production in the process of wound healing of
Calpodes ethilius, in which the precursor of phenol-oxidizing enzyme is activated by an
activator (1966). Since then, properties of phenol-oxidizing enzymes possibly involved
in wound healing or defense reactions were characterized by using samples from
coleopteran (Barrett, 1984a), dipterans (Barrett and Andersen, 1981; Barrett, 1987a, b;
Binnington and Barrett, 1988) and lepidopterans (Barrett, 1984b, 1987b; Aso et al.,
1984; Morgan et al., 1990) (summarized in reviews, Ashida and Yamazaki, 1990,
Andersen, 1990, Barrett, 1991, Sugumaran, 1998 or Andersen, 2010). These enzymes
were distinguished from laccase-type enzymes by their characteristics typical of
tyrosinase-like enzymes, and they were also called “wound phenoloxidase”. Until the
early 90s, it was not clear whether the tyrosinase-type enzymes in cuticle is the same
molecule as the proPO found in hemolymph (Barrett, 1991). Currently it appears that
the protease cascade for proPO activation found in the hemolymph is also present in the
cuticle and that these two enzymes are the same. For instance, in B. mori, genes for
many of the components of proPO cascade (PPAE, GRP, PGRP and proBBAEase) are
expressed not only in internal tissues like hemocytes or fat bodies but also in epidermal
cells (Ochiai et al., 1999; Ochiai and Ashida, 2000; Satoh et al., 1998; Chosa et al.,
personal communication). However, until the middle of 90s, there had been no
information on the amino acid sequences (and their cDNA sequences) of any of proPO
cascade components.

23
 The common problem associated with the analyses of tyrosinase-type enzymes in
insects is maybe their “stickiness”. During the study of proPO cascade in Bombyx
hemolymph, it had been noted that once activated the active PO became to show
stickiness to form high molecular mass complex with surrounding proteins (Ashida and
Dohke, 1980). The complex cannot be dissociated even by treatment with SDS and heat,
making it difficult to isolate PO and to analyze its amino acid sequence. As has been
discussed by Kanost and Gorman (2008), many of early studies of POs were hindered
by this difficulty to purify active POs. When I was a graduate student in Ashida’s
laboratory (1996-2001), I experienced this character of the active PO. In PAGEs
(native-PAGE and SDS-PAGE) of the samples containing active PO from the larval
cuticles of B. mori, the protein patterns were completely smear. I understood that
suppression of proPO activation was really the key for biochemical analysis of this
protein. In such situations, Ashida and Brey showed the presence of proPO in the larval
cuticle of B. mori by performing photometric enzyme assay and western blot with the
specific antibody (Ashida and Brey, 1995). In that study, they extracted cuticle proteins
with acidified buffer containing EDTA and serine protease inhibitor to suppress proPO
activation, since it had been known that in acidified pH, proPO activation cascade
reactions is slower. Also known at that time is the fact that some factors in proPO
cascade require calcium ion for proceeding of the downstream cascade reactions.
Therefore, acidic buffer including EDTA strongly blocked the activation of proPO. In
addition, the extract was separated with CM-Toyoperl (cation-exchange) column into
two fractions, the flow-through fraction with proPO and the adsorbed fraction with the
activating components. In the latter fraction, PPAE is present as inactive precursor form
(proPPAE), but by adding calcium ions, the precursor is activated to active PPAE. By
mixing the fraction of the activated PPAE with the flow-through fraction, the proPO in
the mixture is activated. After the establishment of this extraction procedure with the
activation of proPO suppressed, cuticular proPO was purified (Asano and Ashida,
2001a). The internal amino acid sequences of the cuticular proPO are the same with
those determined in hemolymphal proPO. In northern blotting using the cDNA
fragment, no signal for proPO expression was seen in the samples from epidermal cells
(Ashida and Brey, 1995; Asano and Ashida, 2001). As described above, proPO is
synthesized in a kind of hemocytes (oenocytoides) in B. mori, leading to the hypothesis
that the proPO in the hemolymph is trans-epithelially transported to the cuticle, and this

24
hypothesis was proven (Asano and Ashida, 2001a). During or after transportation,
methionine residues on the surface of proPO are oxidized to methionine sulfoxide
(Asano and Ashida, 2001a, b). By this modification, proPO’s functions (activation by
PPAE or enzymological properties) are not affected.
Before the RNAi study by Arakane et al. (2005), the potential roles of
tyrosinase-type enzymes in cuticle sclerotization had been often discussed, since they
can oxidize the four major catecholic substrates found in insect cuticles, including
NBAD and NADA (Fig. 2) (Asano and Ashida, 2001a; Aso et al., 1985). To understand
the actual contributions of proPOs in cuticle sclerotization, genetic analyses were
performed in two model insects. In T. castaneum, there are two genes for proPOs,
TcTyr-1 and -2, and double knockdown of these genes did not show any abnormalities
in cuticle formation (Arakane et al., 2005). In Drosophila, three proPO genes (proPO1,
2 and 3 corresponding to CG5779, CG8193 and CG2952, respectively) are found in the
genome, and proPO1 and proPO2 are constitutively expressed throughout the stages.
proPO3 expression is very weak in normal development (Asano and Takebuchi, 2009;
Irving et al., 2005; Nam et al., 2008), but the expression is induced in a type of
hemocytes, lamellocytes that differentiate after infection by parasites (Nam et al., 2008).
The deletion mutant of both proPO1 and proPO2 does not show any abnormalities in
cuticle formation (Binggeli et al., 2014). These results indicate clearly that proPOs do
not have important roles in cuticle formation.
In relation with the involvement of phenol-oxidizing enzymes in cuticle
melanogenesis, “granular phenoloxidase (granular PO)” found in the cuticle of black M.
sexta larvae should be mentioned (Hiruma and Riddiford, 1988, 2009). Granular PO
appears in “premelanin granules” of the larval epidermal cells in stage dependent
manner in normal developmental process, and during the synthesis, granular PO is
inactive but is activated upon decline of ecdysone titer (Hiruma and Riddiford, 1988).
This enzyme was distinct from tyrosinase-type enzymes present in cuticle and
hemolymph, which was demonstrated with the antibody raised against granular PO.
The presence of sugar moieties with mannose and sialic acid in granular PO is distinct
from the characters of proPO that does not have sugar chains (Asano and Ashida, 2001a,
b; Yasuhara et al., 1994). The absence of sugar chains in proPOs is important, because
proPOs do not have signal sequence and secretion of proPO to hemolymph is thought to
be achieved by cell rupture of proPO producing hemocytes (Kawabata et al., 1995),

25
implying that proPO is not transported to ER- Golgi apparatus where glycosylation
occurs. The gene for granular PO has yet to be identified, but regulation of the synthesis
and enzymatic activation of granular PO is important subject to reveal molecular
mechanisms of hormone dependent regulation of cuticular pigmentation and also
melanic color pattern formation.
Regarding the relationship between cuticle pigmentation/sclerotization in normal
developmental process and the expression of proPO (and other components of its
activation cascade), a study has shown a possible involvement of proPO in
pigmentation of scales in the swallowtail butterfly, Papilio polytes (Nishikawa et al.,
2013). Expression of the genes for proPO45 and yellow-d2 (and also factors in Toll
pathway such as snake or Späztle-5) are upregulated in red part of the wings where the
yellow-red pigments, papiliochromes, accumulate (papiliochromes are conjugates of
kynurenine and NBAD (Rembold and Umebachi, 1984; Umebachi, 1985). This could
be the first to support the hypothesis that gene for proPO contributes to synthesis of
cuticular components during normal development. It was also shown that toll receptors
and their ligands, spzs, are involved in pigmentation of larval cuticle in B. mori (KonDo
et al., 2017). Spzs are synthesized as inactive precursor form and secreted to
extracellular space but is activated by proteases to become mature ligands that can be
bound by the specific receptors. In the study by KonDo et al., the authors focused on a
mutant that shows black stripe pattern (Zebra: Ze). Among the three genes in the locus
responsible this phenotype, the gene for spätzles-3 (Bmspz-3) was identified as the exact
gene for the phenotype, through the genetic analyses of knocking down and ectopic
expression of this gene. Toll-8 (Bmtoll-8) was also identified as a putative receptor for
Bmspz-3. As described, there is close relationship between proPO activation and
toll-signaling, but there were no data showing the link between proPO activation and
pathway of toll-8 and spz-3 in Ze. Instead, the expression of the gene for yellow is
modulated, with those of the genes for TH, DDC or MCO2 unchanged.

3.2.3 Evolution of type-3 protein in arthropods

As described above of this section, proPO is homologous to arthropod Hcs.
ProPOs and arthropod Hcs belong to type-3 copper protein family. They have two
copper binding sites, CuA and CuB from the N-terminus. Tyrosinases and mollusc Hcs
are also categorized in type-3 copper protein, but the sequences of their CuA sites are

26
partially different from those of proPOs and arthropod Hcs (the position of the second
histidine among the three histidines for copper binding is different between CuAs of
tyrosinase/mollusk Hcs and proPO/arthropod Hcs (Fig. 6)) (summarized by Aguilera et
al. (2013) or Costa-Paiva et al. (2018)). When the cDNA cloning of proPO were
reported, the general understanding in the field was that insects did not have Hcs (Law
and Wells, 1989; Magnum, 1985; Willmer et al., 2000), because insects have well
developed tracheal system. In the observation of Bombyx tissues by immune-gold
labeling, proPO was localized in the tracheal cuticle in high concentrations (Ashida and
Brey, 1995). It was speculated that proPO could have some roles in oxygen
transport/exchange, and this could be an attractive hypothesis in insect physiology.
However, in 1998 a cDNA for putative Hc was isolated from the developing embryos of
the grasshopper, Schistocerca americana (Sànchez et al.) (I myself was very interested
in that hypothesis on the new functions of proPO, and I remember that I introduced the
report of putative Hc cDNA in the seminar of Ashida’s laboratory at that time). Then,
oxygen binding of insect Hc was shown in the study of the stonefly, Perla marginata.
Since then, genes for Hcs were found in multiple hexapods like primitive non-insect
hexapoda (Colembora (springtails) and Diplura (two-pronged bristletails)), non-winged
insects (Archeognatha (jumping bristletails) and Zygentoma (silverfishes)),
hemimetabolous insects (Dermaptera, Orthoptera, Blattodea or Plecoptera), but not in
Odonata (dragonflies), Ephymeroptera (mayflies), Hemiptera or holometabolous orders
like Coleoptera, Lepidoptera or Diptera (summarized by Burmester and Hankeln (2007)
or Pick et al. (2009)). From the phylogenetic analyses, it can be extrapolated that the
arthropod hemocyanin might have evolved from ancestral protein possessing
phenol-oxidizing activity (Burmester, 2001; Holde et al., 2001). Hcs still have potential
to become derivatives showing enzyme activity to oxidize phenolic substances after
structural modifications. Hc from the horseshoe crab (chalicerate), Tachypleus
tridentatus, becomes to be enzymatically active form without proteolysis but by
interaction with factors of coagulation cascade (Nagai and Kawabata, 2000) or by
interaction with small antimicrobial peptide, tachyplesin (Nagai et al., 2001). Hcs from
crustaceans also can be converted to derivatives showing PO activity after treatment
with alcohols or SDS (reviewed in Decker et al., 2007). These characters of Hcs,
together with the observation that Hcs accumulate in crustacean cuticles in high
concentration (Adachi et al., 2005; Kuballa et al., 2008), imply the possibility that Hcs

27
may have some roles in the process of cuticle tanning in crustaceans, though this
hypothesis has not been examined at the levels of molecular genetics. In molecular
phylogenetic analysis of arthropods’ Hcs (Etras et al., 2009), insect Hcs and crustacean
Hcs form isolated clusters with an exception that Hcs of a group of marine crustaceans,
remipedes, the closest crustacean relative of insects, fall into the clusters of insect Hcs,
confirming the close relationship between Insecta (Hexapoda) and Remipedia.

4. Evolution of the genes for MCO2 in arthropods, and possible contributions of

MCO2 in the success of insects as terrestrial animals.

4.1. Evolution of MCO2 in the history of arthropods’ evolution

From this section, I try to describe about relationship between evolution of insects and
that of MCO2, more exactly on a new theory of how MCO2s have contributed to the
success of insects as terrestrial animals. Due to the recent advances of techniques in
obtaining and analyzing numerous amounts of data sets from genomes of multiple
organisms, it has become easier to discuss more precisely the phylogenetic relationship
of taxa within insects or within arthropods. By comparing genomic information, it is
also possible to discuss which genes are shared among arthropods, and to estimate
divergence time between linages. The first insect genome assembly (D. melanogaster)
was published in 2000 (Adams et al.), followed by those of model insects or important
disease vector insects such as A. mellifera (The honeybee genome sequencing
consortium, 2006), T. castaneum (Richards et al., 2008) and A. gambiae (Holt et al.,
2002). Later, the completion of the first genome analyses of chelicerates and
crustaceans was reported (Grbić et al., 2011 and Colbourne et al., 2011, respectively).
Subsequently, reports on the analyses of myriapod genomes were published in 2014 for
Chilopoda (Chipman et al.) and in 2020 for Diplopoda (Qu et al.). The genomic
information of all the four arthropod sub-phyla are now available, and currently it is
widely believed that these arthropods are monophyletic (Fig. 7) (reviewed by Giribet
and Edgecombe (2019) or Schwentner et al. (2017)). The split of the common ancestors
of chalicarates and mandiblates occurred initially, and then the common ancestors of
myriapods and pan-crustaceans diverged. Finally, crustaceans and insects (hexapods)
emerged inside the linage of pan-crustaceans. The latest theory proposes a novel view

28
that is different from that commonly believed. In recent phylogenetic trees, insects are
nested within the cluster of crustaceans, and a taxon of marine crustaceans, Remipedia,
constitutes sister group of hexapods, indicating that Insecta is not a sister group of
Crustacea, but just a subset of Pan-crustacea or that insects represent a terrestrial linage
of crustaceans (Giribet and Edgecombe, 2019; Schwentner et al., 2017; VanHook and
Patel, 2008).
By the recent large-scale analyses of phylogenetic relationship among arthropods,
it is becoming clearer that occurrence of insects (or hexapods) could go back to the
early Ordovician, suggesting that they were one of the earliest animals forming the first
terrestrial ecosystems with plants (Misof et al., 2014). Fossil records have also
supported the early terrestrialization of myriapods and chelicarates (reviewed by
Kenrick et al. (2012) or Selden (2005)). Cuticle must have played advantageous roles
when arthropods started colonization on land environments and away from the sea
water. Cuticle serves as a mechanical support against gravity without support by
surrounding water and as a barrier to prevent dehydration and maintain homeostasis
inside the body cavities. To overcome the difficulties accompanied by colonization of
lands, insects might have evolved physiological systems like tracheal structures
internalized into deep inside the bodies for gas exchange, tightening the sealing in
cuticles to minimize water loss, and external structures like wings for flight and hind
legs for jumping to enable energetically efficient and high velocity locomotion through
the air. Insects also employed the cuticle hardening process that uses catecholamine
oxidation mediated crosslinking that is catalyzed by MCO2. Among insects’
innovations, this MCO2-mediated system could have been one of the most important
traits, because of 1) availability of resources abundant in terrestrial environments and 2)
the physical characteristics of the cuticles made by the MCO2-mediated system. To
discuss possible contributions of MCO2 in the history of insects’ evolution, the time
point when the gene for MCOs was acquired by insects in the history of arthropod
evolution is a point to be considered. To understand the detailed evolutionary history or
phylogenetic positions of MCO2s, molecular phylogenetic analyses were performed
(Asano et al., 2014, 2019; Yue et al., 2019). In the report of biochemical
characterization of BmMCO2 precursor, a phylogenetic tree was constructed to know
the position of insect MCO2s in the evolution of metazoan 3dMCOs (Asano et al.,
2014). In that tree, 3dMCO-like sequences were collected from various phyla and

29
sub-phyla including Nematoda, Mollusca, Urochordata, Echinodermata,
Cephalochordata, Crustacea, Myriapoda and Insecta. The proteins are divided into two
clusters, one having proteins with Cys-rich region that had been identified in the report of
the first cDNA cloning for 3dMCOs from M. sexta and A. gambiae (Dittmer et al., 2004),
and the other having proteins without the Cys-rich region. Inside the cluster of the
proteins with the Cys-rich region, there are two sub clusters, one with MCO1s and the
other with MCO2s. In the cluster with insect MCO1, insect MCO1s and proteins from
non-insect arthropods constitute a cluster isolated from non-arthropod proteins,
indicating the close relationship between MCO1s and the proteins of non-insect
arthropods (MCO1-related proteins). In the cluster with MCO2, only insect proteins are
included, indicating that MCO2 is a protein that evolved independently from molecular
evolution of proteins in the cluster of MCO1s and MCO1-related proteins.
After the report in 2014, a review on insect terrestrialization was published in 2019
(Asano et al.), and in that review a new phylogenetic tree of 3dMCOs was constructed to
further understand the position of insect MCO2s in the evolutionary history of
arthropods’ 3dMCOs. For making this tree, all the available sequences at that time from
non-insect arthropods were collected together with sequences from multiple orders of
insects, covering both holo- and hemi-metabolous insects like Odonanta, Orthoptera,
Hemiptra, Coleoptra, Lepidoptera, or Diptera. Fig. 8 is the molecular phylogenetic tree of
arthropods’ 3dMCOs constructed with sequences from representative insect species and
those of non-insect arthropods (in this tree, like the tree in Asano et al., 2014, there are no
sequences from chelicerates, because in genomic databases like “Emsemble metazosa” or
“i5k”, genes for 3dMCOs were not found in chelicerates). The new tree may explain the
process of 3dMCOs’ evolution in arthropods more in detail, because the new tree
includes proteins from multiple orders of Crustacea (Isopoda, Decapoda and
Malacostraca) and non-insect Hexapoda (Colembora and Diplura) that could not be
included in the old tree. The phylogenetic tree in Fig. 8 is divided into two main clusters,
one with MCO1s (and MCO1-related proteins) (=cluster A) and the other with MCO2s
(=cluster B). In cluster A, there are proteins of non-insect arthropods (myriapods,
crustaceans and non-insect hexapods). In contrast, in cluster B only insect proteins are
included, suggesting that the ancestral protein of MCO2s might have emerged after
divergence of insects from the common ancestors with non-insect hexapods. In cluster B,
the lengths of branches in the sub-cluster of MCO2s (indicated with darker box) are very

30
short, indicating the highly conserved sequences among MCO2s, which has already been
pointed out previously (Dittmer et al., 2004). It can be speculated that only minute
mutations in protein sequence may affect greatly to MCO2 functions or regulations of the
molecular functions. Regarding the time point when insects acquired MCO2 gene, any of
non-insect arthropods including the closest relatives of insects, springtails (Collembora)
and two-pronged bristletail (Diplura), do not have genes for MCO2s. The oldest extant
winged insects (Odonanta and Ephemeroptera) have proteins (EDAN008795-PA and
LFUL009665-PA, respectively) that fall into the cluster with MCO2s. Because of
shortages of information in open databases, it is unknown whether the primitive
non-winged insects (jumping bristletail and silver fish) have genes for MCO2s. To obtain
answer to this question, it is required to check the presence of MCO2 genes in the two
insect groups.

4.2 MCOs in insects: MCO1s, MCO-related proteins and others

In the above sections, molecular properties, and gene functions of MCO2s have been
mainly described with the reason that this review focuses on MCO2 and its functions in
cuticle physiology. In the current section, focus will be on MCOs other than MCO2 and
their importance to insect physiology.
MCO1s are very important, because genes for MCO1s are necessary for a variety of
physiological processes in insects (Lang et al., 2012; Peng et al., 2015), and also
non-insect arthropods (except chelicerates) have proteins with high homology to insect
MCO1s (MCO1-related proteins) (Asano et al., 2014, 2019), implying some conserved
functions. Lang et al (2012) reported the molecular properties of insect MCO1s first using
recombinant MCO1 of D. melanogaster (DmMCO1). Among the three Drosophila
proteins investigated (DmMCO1, DmMCO2A and DmMCO3), DmMCO1 solely had
ferroxidase activity, though all the three proteins showed activity to laccase substrate like
p-phenylenediamine. The presence of ferroxidase activity is consistent with the
conservations of iron-binding acidic residues between yeast ferroxidase (Fet3p) and
insect MCO1s. From the observations that DmMCO1 knocked-down individuals showed
lower iron accumulation or that MCO1 protein was detected in the basal surfaces of
digestive tubes or Malpighian tubes, it is thought that MCO1 is involved in iron
homeostasis. The strong knockdown of DmMCO1 leads to lethality in pupal stage,
indicating the important roles of this gene in development, probably through the

31
ferroxidase activity. Molecular and gene functions of MCO1s were also studied using M.
sexta and A. gambiae. In these insects, MCO1 proteins locate at the surface of the midgut
cells facing to hemolymph, which is the same with the observation in D. melanogaster.
The recombinant MCO1 proteins (MsMCO1, AgMCO1 and TcMCO1) have similar
enzymatic properties with those of DmMCO1 (Peng et al., 2014), and it was also shown
that all the recombinant proteins have strong oxidase activity against ascorbate, compared
to those against ferrous ion and diphenols, suggesting that insect MCO1s are involved in
iron metabolisms through ascorbate oxidase activity. In these analyses, the data did not
indicate involvement of MCO1s in cuticle pigmentation and sclerotization, which is
consistent with the result of the previous study that knockdown of the gene for MCO1 in
T. castaneum (Arakane et al., 2005). Unlike MCO2s, MCO1s and MCO1-related proteins
are found widely in arthropods except chelicerates, suggesting some common roles in
arthropods (except chelicerates). It is possible that like MCO1s of insects, the
MCO1-related proteins in non-insect arthropods may function through activities of
ferroxidase or ascorbate oxidase.
In 2015, there was a report on the genes for MCO-related proteins (MCORPs) that
are highly homologous to insect MCOs (Peng et al., 2015). They are present in variety of
insect orders. Their notable characteristics is the lack of residues required for
coordination of copper atoms. The recombinant MCORP of T. castaneum (TcMCORP)
contained very small amount of copper atoms and showed no absorbance peaks in
spectrophotometry that are characteristic of copper proteins. In A. gambiae, the gene for
MCORP is expressed in many tissues, and the MCORP knockdown in T. castaneum
resulted in mortality or suppression of oocyte maturation.
Other than MCO1, MCO2 and MCORP, molecular characterization of a mosquito
specific MCO was reported (Lang et al., 2012). MCO3 of the A. gambiae were
synthesized in baculovirus system, and the recombinant protein (AgMCO3) showed
activity against laccase substrate (ABTS), but not against substrate for ferroxidase. This
protein is seen at the space between blood meal and midgut epithelium, indicating that
this protein is localized in peritrophic membrane. There is another example of studies on
genes for MCOs. Molecular characterization of enzyme showing laccase-type activity in
the salivary glands of the rice leafhopper, Nephotettix cincticeps was reported (Hattori et
al. 2005). This finding was the first to show laccase-type activity in salivary glands of
insects, and the authors hypothesized that the enzyme responsible for the laccase-type

32
activity in the salivary glands might be involved in metabolizing substances in rice plants.
Later, cDNAs for 3dMCOs were identified in this species, and it was hypothesized that an
N. cincticeps ortholog of MCO1 (NcMCO1S) would be responsible for the laccase-type
activity (Hattori et al., 2010). In the recent analysis, it was concluded that NcMCO3 is
probably the gene for the laccase-type activity in salivary glands. Though NcMCO1S is
surely expressed in the salivary gland, knockdown of not NcMCO1S but NcMCO3
resulted in disappearance of laccase-type activity in salivary glands (Matsumoto and
Hattori, 2019).

4.3 Possible advantages of MCO2-mediated system in colonization of land

The importance and possible contribution of MCO2-mediated systems to successful
terrestrialization of insects has been previously proposed by our group (Asano et al., 2014,
2019). To understand this hypothesis, it is important to know the difference in the
systems for cuticle hardening among arthropods, especially the difference between
insects and crustaceans, because they are closely related taxa, and more precisely Insecta
is a taxon diverged from a linage of Crustacea. Insects are characterized by the extreme
diversity, the huge biomass, and their large impacts on ecosystems. They are also
regarded as the dominant animals in terrestrial environments. Like insects as terrestrial
animals, crustaceans arguably are the most abundant group of marine animals
(Schwentner et al, 2017). Crustaceans are also morphologically the most diverse among
any taxa (Martin and Davice, 2001), even though they have comparatively fewer species
than insects. It is assumed that both insects and crustaceans have high potentials to adapt
to various environments by diversification. However, why are the main habitats of insects
and crustaceans different (terrestrial and marine, respectively)? To answer this question,
one has to look into the mechanisms that attribute success for survival of these two animal
systems. A major difference that can be immediately recognized is the way in which
these two animal systems protect themselves from the environment viz, cuticular
hardening. It is well known that crustaceans accumulate calcium ions for cuticle
hardening. In the marine environment, calcium ion can be easily obtained from the
surrounding sea water (Deshimaru et al., 1978). But when they start colonizing in the
terrestrial environments (either in fresh water or on land), they might have met with a
difficulty in calcium supply that is abundant in the marine environment. To overcome this

33
difficulty, some decapods evolved efficient calcium transporter protein that can provide
higher absorption potential for calcium ions from fresh water than those of marine species.
They also make crystals of calcium carbonate (gastroliths) using calcium ions from the
old cuticle before molt, and the calcium ions are re-used to harden the new cuticle. In
some species of terrestrial isopods, ecdysis is done in two steps (biphasic manner), which
is a system to restrict loss of calcium. In the first step, the posterior part of the cuticle is
shed, but prior to molting, the calcium ions in the old cuticle have already moved to
anterior part of the body (between the cuticle and the hypodermis). Then, the old cuticle
of the anterior part is shed, but the calcium ions have already been transported to the
posterior new cuticle before shedding (these strategies of crustaceans are reviewed by
Luquet (2012) or Wheatly (1999)). In some species, the calcium ions in exuviae are also
consumed by eating (Steel CGH. 1993; Ziegler et al., 2007). Daphnia is a genus of small
planktonic crustaceans that are classified into Branchiopoda. It is known that majority of
the branchiopod species are living mainly in fresh water. One hypothesis states that
branchiopods are the closest extant relatives of insects/hexapods, while another
hypothesis advocates that remipede, a group of marine crustaceans, is the closest extant
relative of insects/hexapods (Giribet and Edgecombe, 2019; Schwentner et al., 2017). It
has been shown in Daphnia magna that 85-90 % of total calcium storage is lost during
molting even in the environment of low calcium concentrations (Porcella et al., 1969;
Alstad et al., 1999). It was also shown that in low calcium conditions, Daphnia pulex
could not increase body size, extend neck spine, and harden carapace (Riessen et al.,
2012), all of which are defense responses after perception of substances from predators.
These observations indicate positive correlation between calcium contents and cuticle
formation/hardening in this genus. All the information described indicates that after
colonization of terrestrial environments, crustaceans have continued to utilize calcium
ions to harden their cuticles. In contrast to crustaceans, insects evolved the
MCO2-mediated system that does not require high levels of calcium supply. The
MCO2-madiated system utilizes oxidation reactions of catecholamines, but as depicted in
Fig. 9A, this process requires consumption of molecular oxygens as acceptors of the
electrons from the substrates. In reactions of catecholamine oxidation, four electrons
from two substrates are transferred to a molecular oxygen, and the final products are two
quinones and two water molecules. In the period when the ancestors of insects (or
hexapods) colonized on land (early Ordovician (Misof et al., 2014)), the content of

34
oxygen in the atmosphere had reached to the levels comparable to the current value
(reviewed in Hsia et al., 2013). Because of its low solubility in water, the content of
molecular oxygen in water is much lower, compared to the level in the atmosphere
(30-fold). In addition, viscosity and density of air are much lower than those of water (50-
and 1000-fold, respectively). With these factors, absorption of oxygen from the air is
much easier than from the water (Fig. 9B). The MCO2-mediated reactions with high
oxygen consumption might have evolved in accordance with colonization into
atmospheric conditions rather than in water (Asano et al., 2019). The MCO2-mediated
system provided insects opportunities to go into inland areas much more freely than
animals requiring calcium ions for construction of body structures. During the expansion
of territories, the early insects might have been one of dominant animals occupying the
new niches in the primitive terrestrial ecosystems, with their high potentials of adaptation
by diversification. It is well accepted that insects were the animal first to fly in the history
of metazoan. By acquiring the MCO2-mediated system, insects were able to harden their
cuticles without high levels of biomineralization that leads to increasing of body weights.
This character of insect cuticle (light but strong) could be one of the critical factors
providing insects the ability to fly. It is no doubt that flight ability is very advantageous in
survival competitions, which can be explained with the fact that over 99% of the
described insect species are winged. Even without wings, it might be possible that insects
could be one of dominant animals in early terrestrial ecosystems after acquiring the
MCO2-mediated system. Currently it is thought that the evolution of flight might have
started from gliding from high places (Dudley et al., 2007; Ross, 2010). It can be
speculated that the early non-winged insects might have been dominant sufficient to
occupy niches in high places like tall trees for repetitive trials of gliding until they finally
acquired the ability of gliding. With these speculations, the possible impacts of
MCO2-mediated system on the evolutionary history of insects seem to be considerably
high, and without this system, the evolution of insects might have become very different
from what we see now.

4.4 Candidates of enzymes that substitute functions of MCO2

MCO2-mediated system might have evolved specifically in insects, because MCO2 is
insect specific (Asano et al., 2014, 2019). However, because MCO2 is not the only
protein to catalyze catecholamine oxidation, systems like MCO2-mediated system could

35
exist in non-insect arthropods. MCO1s can oxidize broad ranges of substrates, for
instance, MCO1s have activity against substrates for laccase, though enzymological
properties of MCO1s are not optimized for diphenol oxidation (Lang et., 2012; Peng et al.,
2014). It is possible that MCO1-related proteins in non-insect arthropods (Fig. 5) have the
similar enzymological properties as that of MCO1s and that they are candidates of
enzymes substituting the functions of MCO2. Genes for POs are found widely in
arthropods except chelicerates (Scherbaum et al., 2018), and there are also candidates of
enzymes capable of substituting MCO2 functions. Chelicerates do not seem to have POs,
but instead, possess Hcs that could function as POs as shown in vitro experiments. Hcs
can be converted to derivatives showing PO activity by interactions with small peptides
or treatment with organic solvents probably to induce conformational changes (Decker et
al., 2007; Nagai and Kawabata, 2000; Nagai et al., 2001). As summarized previously
(Stevenson, 1985), it was shown that diphenol-oxidizing activity was detected in the
cuticle of decapod crustaceans such as Cancer pagurus, Orconectes obscurus,
Clibanarius olivaceous or Carcinus maenas (Dennell, 1947; Stevenson and Adomako,
1967; Chockalingham, 1974), though it was not known whether the activities found in
these species were categorized to MCO-type or PO (Hc)-type. Catechols found in insect
cuticle, NADA and N-acetyl-noradrenalin (N-acetyl-norepinephrine: NANE), were
identified in the cuticles of the fiddler crabs, Uca pugnax and U. pagilator (Vacca and
Fingerman, 1972; Summer, 1967). In U. pugnax, it was demonstrated that NADA was
synthesized in isolated epidermal cells (Summer, 1968). Therefore, it is possible that
these animals could use sclerotization process using enzymes substituting MCO2s
(MCO1s, POs and Hc derivatives) as described for insects, though no derivatives of
catecholamines typically found in sclerotized insect cuticles were identified in the
cuticular hydrolysates of these animals (Andersen, 2010).
To further understand the origin of MCO2-mediated system, one must consider not
only the evolution of MCO2, but also the factors involved in synthesis of
MCO2-substrates. It is interesting that the genes for TH and DDC of insects express
epidermal cells’ specific isoforms, which may be an adaptive trait for proper regulation of
cuticle hardening and pigmentation (Ninomiya et al., 2007; Shen et al., 1993; Vie et al.,
1999). TH and DDC are widely distributed in metazoans as they are necessary to produce
DA, the neurotransmitter. Once DA is generated, then one must look for dopamine
modifying enzymes, aaNAT or NBADS in non-insect arthropods. Previously it was

36
hypothesized that the ancestor of insects might have evolved accumulation of phenolic
compounds in the cuticle (Asano et al., 2019), based on an assumption that phenolic
compounds can absorb UV for protection from strong sunlight in the atmospheric
conditions. In addition, NBAD has antimicrobial activity and epidermal expression of the
gene for NBADS is induced by microbial infection, indicating that NBAD actively
participates in immune response in integuments (Scachter et al., 2009). It is possible that
phenolic compounds used for these purposes might have been diverted to substrates of
MCOs for cuticle sclerotization.
Although both MCOs and POs can catalyze oxidation of o-diphenols to produce
o-quinones, it is not known why not POs but MCO2s are used for cuticle sclerotization.
An idea to answer to this question is the difference in the catalytic mechanisms. Type-3
copper proteins like tyrosinase remove electron pair from hydroxyl groups of o-diphenols
to produce o-quinones, and 3dMCOs remove one electron from the substrate and the
product is o-semiquinone, a radical cation (Solomon et al., 1995). In the latter case, as
secondary reactions, two-electron oxidized products (quinones) are produced by
disproportionation of the radicals that is accompanied by production of the parent
substrates (reviewed by Manzano-Nicolas et al. (2020), Solomon et al. (1995) or
Sugumaran (2016)). In reviews in industrial application using these copper oxidases, it is
mentioned that laccase-mediated crosslink formation is based by formation of radicals,
which, in turn, can further react with other phenolic radicals or free aromatic or amino
groups in proteins (Buchert et al., 2010). In plants, the phenotypic characterizations of
Arabidopsis thaliana have shown that the genes for laccases and peroxidases are
intimately associated with lignin formation. Studies also confirm that the biosynthesis of
lignin occurs through formations of free radicals that are catalyzed by laccase and
peroxidase (Zhao et al., 2013; reviewed by Agustin et al. (2021) or Tobimatsu and
Schuetz (2019)). Lignin is one of the major cell-wall components (estimated to make up
20-35% of the dry weight of woods (Li, 2011)), which is produced by oxidative
polymerization of lignin monomers (phenyl propanes). Lignin is thought to be an
innovation of terrestrial plants that might have evolved when the level of atmospheric
oxygen was high (Willis and McElwain, 2002). Though plants have both tyrosinase-type
oxidases (polyphenoloxidases) and 3dMCOs including laccase (Pretzler and Rompel,
2018), plants use laccases and peroxidase to oxidize lignin monomers for production of
radicals. It is known that peroxidases facilitate oxidation of phenolic substances to

37
produce corresponding phenoxy radicals. During lignin polymerization, quinone
methides are produced through disproportionation of one electron oxidized products from
lignin monomers (lignin phenoxy radicals) or oxidation of lignin phenoxy radical
preceded by proton detachment (reviewed in Ralph et al., 2009; More et al., 2021). In
insects also production of quinone methides is one of key steps in cuticle sclerotization.
Quinone methides can be produced during oxidation of o-diphenols in the reactions
catalyzed by partially purified trypsin-solubilized laccase-like enzyme from M. sexta
(Thomas et al., 1989). Production of p-quinone methide is easily associated (up to 40% of
the total products) with oxidation reactions mediated by the Manduca enzyme, in contrast
to tyrosinase-mediated reactions in which much smaller amount of p-quinone methide
was detected. In that analysis, production of NBANE was measured as the indicator for
production of quinone methides, since NBAD-quinone methide is readily hydrated to the
norepinephrine derivative (reviewed in Andersen, 2010; Sugumaran, 2010). There is
another route for quinone methide production that is catalyzed by quinone isomerases.
Quinone isomerases were purified from hemolymph of S. bulluta (Saul and Sugumaran,
1990) and from larval cuticles of H. cecropia (Andersen, 1989). It is possible that the
Manduca enzyme preparations contained both MCO2 and quinone isomerase, resulting
the high ratio of NBANE detection. However, irrespective of this discussion on the purity,
it is worth considering that radicals probably produced in MCO2-mediated reactions are
directly used for making crosslinks in insect cuticle, though participation of radicals has
been less discussed, compared to the studies in plants. Both insects and plants adopted
MCO-mediated (and peroxidase-mediated) reactions to synthesize structural components
with mechanically excellent properties. These systems might have been evolved/selected
as traits to utilize high availability of molecular oxygen present in the atmosphere, which
could be an example of convergent evolution in molecular processes.

4.5 Adaptation to terrestrial environments by MCO2-mediated system and the

reason why insects are rare in marine ecosystems
In this section, the possible contributions of MCO2-mediated system in the evolutionary
history of insects have been described, but it must be kept in mind that insects have
evolved other traits such as complete metamorphosis, eusociality, symbiosis or
co-evolution with plants, all of which have been the topics of active discussions on how
insects have successfully expanded their territories and become so diverse. Arthropods of

38
all four major sub-phyla succeeded in colonizing terrestrial environments, irrespective of
the presence of MCO2. However, considering the possible advantages brought by
MCO2-mediated system, contribution of this system during the adaptation of insects to
terrestrial environments seems to be very high. In summary, the scheme of insects’
terrestrialization that had been presented in the previous review (Asano et al., 2019) is
reconstructed with minor modifications (Fig. 10A), 1) the ancestor of insects/hexapods
colonized on land, 2) on land the MCO2-mediated system might have been selected as a
system to utilize the high availability of molecular oxygen in the air. 3) This might have
been advantageous trait to expand their territories in inland areas poor in calcium, and it is
possible that primitive insects might have been dominant to some extent in primitive
terrestrial environments, 4) MCO2-mediated system without bio-mineralization provided
light but strong cuticle that might have been one of critical factors enabling insects to fly.
Insects were the first animal to fly, which might have been very advantageous for
acceleration of their expansion of the habitats, accompanied by creation of new niches
that could not be accessed by the competitors.
At the end, I try to propose an explanation to a long-lasting discussion on the reasons
why insects are very rare in marine environments, which is very strange considering their
extremely successful positions in terrestrial environments. Many of marine insects are
living in intertidal zones, spending only a part (eggs, larvae or pupae) of their lives
underwater (Cheng, 1976). The only pelagic insects are sea skaters that can complete
their life cycle in ocean environments (Andersen, 1999; Ikawa et al., 2012). However,
they do not live underwater, but on the surface of the seawater. Recently, there is a
proposal that advocates seal lice as the only truly marine insects (Leonardi et al., 2022).
The Seal lice is classified in the order, Phthiraptera, and this group is known as the only
insects that have become obligate and permanent parasites throughout their entire life
cycle. Interestingly, the structure of the phylogenetic tree of seal lice is the same with that
of their host animals (river otter, fur seal, sea lion and seals), indicating that seal lice have
kept the parasite-host relationship since its establishment when the ancestors of seals had
lived on land (Kim, 1998). The high resistance against high hydrostatic pressure or the
gas exchange through the thin part of the cuticle that does not use tracheal systems are the
adaptive traits to survive on deep-diving seals, but the egg and 1st instar nymph cannot
survive when immersed in sea water, consistent with observations that reproduction and
transmission of the seal lice can only occur when their hosts are on land (summarized by

39
Leonardi et al. (2022)). Apart from the discussions on sea skaters and seal lices, there
have been discussions on factors possibly limiting insects to colonize into seawater. The
factors are, for instance, lower resistance against high osmotic pressure caused by salt,
low efficiency of respiratory system for gas exchange in water, presence of predators like
fishes (Harrison et al., 2012; Madderell, 1998). One of the well-discussed hypothesis is
the occupation of marine environments by crustaceans (there are no remaining
niches/open spaces for other arthropods). It seems that this idea from ecological
viewpoint has not been well-explained or supported in the levels of physiology or
molecular evolution, but when the MCO2-mediated system is taken into consideration, it
would be possible to provide a simple explanation. For cuticle formation, insects and
crustaceans utilize different natural resources, molecular oxygen and calcium ions,
respectively, that are abundant in terrestrial (air) and marine (sea water) environments,
respectively. In the sea water, insects cannot harden their cuticles efficiently because of
shortages in molecular oxygen caused by the low solubility, whereas the availability of
plenty of calcium ion is advantageous for crustaceans to keep their ecological positions in
marine environments. In fresh water, the contents of both oxygen and calcium are much
lower than those in the air and the sea water, respectively, hence they can co-exist (insects
cannot exclude crustaceans and vice versa).

5. Concluding remarks
This chapter focused on the recent progresses made on the study of cuticular enzymes
with diphenol-oxidizing activity. Whether tyrosinase- or laccase-type enzyme is
responsible for cuticle hardening used to be an important unresolved question until 90s.
However, research progress made in the last two decades, clearly established the key role
played by MCO2 not only in cuticular hardening reaction but also in cuticle
melanogenesis during development. The insect specific MCO2-mediated hardening
process can be considered as a unique process that offered selective advantage for the
insects, as MCO2-mediated cuticle hardening does not require high calcium supply,
which might have been advantageous during terrestrialization of insects. The light but
strong cuticle achieved by MCO2-mediated crosslinking and hardening of the cuticle
could be a critical factor for the innovation of insects’ flight ability. The new theory on
the contributions of the MCO2-mediated system also proposes a novel and simple

40
explanation to the long-lasting discussion regarding the mystery that only a few insects
are living in marine ecosystems.
The development of molecular biological methods to analyze gene functions (RNAi
or genome editing) and that of methodology for sequence analyses (next generation
sequencing and bio-informatics) have contributed very much to the progress in the field
of insect cuticle science, but there are newly emerged questions, such as molecular
mechanisms for regulation of MCO2 functions, evolution of MCO2-mediated system in
non-winged insects, or programs for proper cuticle formation that may require
orchestration of sub-programs for both behaviors and metabolic pathways. I hope the new
questions will be solved through future studies, and our understanding of cuticle
formation will become much deeper.

6. Acknowledgements
I thank Dr. Manickam Sugumaran for the invitation to contribute to this series, Dr.
Yasuyuki Arakane for the good photos of the beetle (Fig. 1) and deep discussions on
cuticle physiology, Dr. Svend Olav Andersen for the discussion when I visited to his
office in Royal Danish Academy (2011) and also for giving me a volume of Advances in
Insect Physiology (with his signature), Dr. Michael Kanost for his generously accepting
me to spend a half year in his laboratory (2013-2014) (I really enjoyed doing experiments
of MCO2 there), Dr. Masaaki Ashida for directing me to do researches on insect cuticle in
his laboratory (1996-2001), Dr. Keiji Shikama for my first experience to analyze/purify
proteins with oxygen affinity (myoglobins from bovine and sperm whale) (1995-1996),
Dr. Michael Locke for a picture, in which he and I were taken together when we attended
ICE2000 in Iguass falls (2000), Dr. Klaus Reinhardt for encouraging me to publish the
MCO2-related theory (Asano et la., 2019) when I attended the European meeting of
arthropod cuticle at Dresden (2015), Dr. Yosuke Seto, Dr. Kosei Hashimoto, Dr. Hiroaki
Kurushima and Dr. Craig R. Everroad for discussions during building of the
MCO2-realted theory.

Figure legends
Figure 1. Metabolic pathway for cuticle pigmentation (A) and sclerotization (B).

41
This scheme was constructed according to the references (Arakane et al., 2009, Andersen,
2010 and Barek et al., 2022). The factors, the gene of which have been identified, are
indicated with light blue boxes. The four major substrates of MCO2 and PO are indicated
with red-lined boxes. Red arrows indicate oxidation reactions catalyzed by MCO2 or PO.
The photos of T. castaneum were provided by courtesy of Dr. Yasuyuki Arakane

Figure 2. Structures of insect 3dMCOs. (A) Schematic structures 3dMCOs of the lacquer
tree and insects. (B) Schematic structures of alternatively spliced isoforms of BmMCO2.
The figures in the references (Yatsu and Asano, 2009; Asano et al., 2014) are modified.

Figure 3. in vitro activation of BmMCO2 precursor.

In both SDS-PAGE (left panel) and native-PAGEs (middle and right panels) of the
purified BmMCO2 precursor, the sample applied in the right lanes were pretreated with
trypsin. The native-PAGE gels were pretreated with (middle panel) or without (right
panel) isopropanol before staining with DA (Asano et al., 2014).

Figure 4. Analyses of lepidopteran MCO2s. Schematic structures of proteins that have

analyzed in the reports of 2009 (Asano et al., 2014 and Dittmer et al., 2009) are shown.
(B) the graphs were drawn according to the data in the two references. The left graph
shows the data obtained in the conditions of 0.5 mM of DA and pH 6.5 of the reaction
mixtures (Asano et al., 2014). The right graph shows the values of the enzyme activity in
assay conditions of 0.25 mM substrates and pH 6.0 (Dittmer et al., 2009)

Figure 5. Eclosions of the large brown cicada, Graptopsaltria nigrofuscata (A). In B, the
concept of programs for cuticle hardening is drawn. The cuticles of the legs are hardened
at first, then wings are expanded. After completion of wing expansion, cuticle hardening
and pigmentation start in whole body. The orders of behavior and cuticle hardening seem
to be strictly programmed/controlled in eclosion process. The hardness of the cuticles are
indicated by intensities of purple color.

Figure 6. evolution of type-3 copper protein. The figure of the reference (van-Holde et al.,
2001) was modified.

42
Figure 7. Phylogenetic tree of arthropods. The tree of four sub-phyla of arthropods (the
figures in Hughes and Kaufman, 2002 and Asano et al., 2019 were modified). The lengths
of the branches do not have meanings in this tree.

Figure 8. evolution of 3dMCOs in arthropods (the figure in the reference (Asano et al.,
2019) was modified).

Figure 9. Importance of molecular oxygen in the oxidation reactions occurring in insect

cuticle, and high availability of molecular oxygen in terrestrial environments.

Figure 10. The scheme of how MCO2-mediated system affect insects’ habitats.
(A) schematic drawing of insect terrestrialization (modified from Fig.4 of Asano et al.,
2019). (B) schematic drawing of habitat segregation between sea skaters and marine
crustaceans.

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