432
12 Small Nitrogenous Compounds
OH
o
CD/ 0 "--..0 OH
a)
c9r oH b)
II
0 '
~c9roH
0
7
9H2
0
,0
CH 2
R
~HCOCH3
CH
l
II
CH
I
c9r
oH OH
CD rCOCH3
OH
NH-Protein
~OH
reD
~
k
OH
Protein -NH~NH-protein
R
HC-Protein
I
HC-Protein
~HCOCH3
I
Fig. 12.12a, b. Sclerotization of insect cuticulae [4, 138]. a
Quinone tanning. N-acetyldopamine (NADA) is oxidized
to the corresponding quinone by phenoloxidase and binds
to protein; the protein-catechol complex is further oxidized
to quinone and is linked to a further polypeptide by
a bridge of incompletely defined structure. b ~-sclerotization. Protein chains are bound to the ~ and a carbon
atoms of the NADA side-chain; various ~-keto compounds
are produced from these bridge structures by hydrolysis.
The ~-sclerotization probably involves an intermediate
with an unsaturated side-chain. 1, Phenoloxidase; 2, Nacetyldopamine desaturase
NADA; however, this is probably not the result
of direct de saturation of NADA but is due to the
tautomerization of NADA-o-quinone methide.
Dehydro-NADA-quinone can form benzodioxin
dimers [6, 215,238]. It is completely feasible that
ring and side-chain bonds arise on the same catechol molecule so that the bridge structure has a
protein: catechol ratio of 4: 1 or 3: 1 instead of 2: 1
[138].
The ratio of quinone tanning to ~-sclerotization differs markedly with the species and
developmental stage, e.g. in the pupa of the lepidopteran Manduca sexta it is 75:25, in the puparium of the fly Sarcophaga bullata it is 85: 15 and
in the adults it is 97:3, in the ootheca of the cockroach Periplaneta americana it is 91: 9, whilst in
the cuticula of the adults it is 17:83, and in the
femur of the migratory locust Locusta migratoria
it is 10:90 [4]. There is some correlation between
the type of bridge and the colour of the cuticula;
in weakly pigmented skeletal structures one finds
mainly ~-sclerotization of the side-chains of catecholamine, and in darkly coloured structures
there exists mainly quinone bridges, but the ratio
of the two bridge types can vary widely even in
dark regions of the cuticula [4, 157]. The degree
of pigmentation of the cuticula is probably greatly
dependent upon the spectrum of the acy1catecholamines, which serve as substrates for the oxidative enzymes. Thus, for example, the sclerotized,
transparent cuticula of Manduca sexta and other
insects contains mainly N-acetyldopamine but the
hard, dark brown regions have mainly N-~alanyldopamine [128].
Many insect cuticulae contain fIrmly bound IJalanine. The incorporation of this amino acid into
the cuticula has been examined in detail for the
puparium of flies in which the haemolymph of larvae just prior to pupation contains large quantities of free ~-alanine or ~-alanyl dipeptides [138].
During sclerotization, the ~-alanine is incorporated into the cuticula. This process is disturbed
in some mutants of the fly Drosophila melanogaster and the beetle Tribolium castaneum [210]. The
Drosophila mutant "black" is unable to produce
~-alanine, and the mutant "ebony" cannot incorporate it into the epidermis; in both cases, the
cuticula of the adult animal is black instead of
brown. In isolated cuticulae, dopamine alone
produces a black colour, whereas N-~alanyldopamine gives the normal brown colour.
Thus, ~-alanine apparently blocks the amino
group of dopamine, inhibits melanin production,
and promotes the formation of sclerotizing structures from catecholamine [4, 118]. The localization and the chemical structure of ~-alanine
bonds in sclerotized cuticulae are not known
[4, 138]. It is also not clear whether, and in what
way, the differences in catechol spectra between
different insect species and between different
regions of the cuticula influence the process of
sclerotization and the properties of the sclerotized cuticula [157].
Compared with sclerotization of the insect cuticula, the hardening of the crustacean shell has
been relatively little investigated [23]. In the fiddler crab Uca pugilator, dopamine is converted to
N -acetyldopamine and incorporated into cuticular proteins of the post-moulting animal. In con-
12 Small Nitrogenous Compounds
OH
o
CD/ 0 "--..0 OH
a)
c9r oH b)
II
0 '
~c9roH
0
7
9H2
0
,0
CH 2
R
~HCOCH3
CH
l
II
CH
I
c9r
oH OH
CD rCOCH3
OH
NH-Protein
~OH
reD
~
k
OH
Protein -NH~NH-protein
R
HC-Protein
I
HC-Protein
~HCOCH3
I
Fig. 12.12a, b. Sclerotization of insect cuticulae [4, 138]. a
Quinone tanning. N-acetyldopamine (NADA) is oxidized
to the corresponding quinone by phenoloxidase and binds
to protein; the protein-catechol complex is further oxidized
to quinone and is linked to a further polypeptide by
a bridge of incompletely defined structure. b ~-sclerotization. Protein chains are bound to the ~ and a carbon
atoms of the NADA side-chain; various ~-keto compounds
are produced from these bridge structures by hydrolysis.
The ~-sclerotization probably involves an intermediate
with an unsaturated side-chain. 1, Phenoloxidase; 2, Nacetyldopamine desaturase
NADA; however, this is probably not the result
of direct de saturation of NADA but is due to the
tautomerization of NADA-o-quinone methide.
Dehydro-NADA-quinone can form benzodioxin
dimers [6, 215,238]. It is completely feasible that
ring and side-chain bonds arise on the same catechol molecule so that the bridge structure has a
protein: catechol ratio of 4: 1 or 3: 1 instead of 2: 1
[138].
The ratio of quinone tanning to ~-sclerotization differs markedly with the species and
developmental stage, e.g. in the pupa of the lepidopteran Manduca sexta it is 75:25, in the puparium of the fly Sarcophaga bullata it is 85: 15 and
in the adults it is 97:3, in the ootheca of the cockroach Periplaneta americana it is 91: 9, whilst in
the cuticula of the adults it is 17:83, and in the
femur of the migratory locust Locusta migratoria
it is 10:90 [4]. There is some correlation between
the type of bridge and the colour of the cuticula;
in weakly pigmented skeletal structures one finds
mainly ~-sclerotization of the side-chains of catecholamine, and in darkly coloured structures
there exists mainly quinone bridges, but the ratio
of the two bridge types can vary widely even in
dark regions of the cuticula [4, 157]. The degree
of pigmentation of the cuticula is probably greatly
dependent upon the spectrum of the acy1catecholamines, which serve as substrates for the oxidative enzymes. Thus, for example, the sclerotized,
transparent cuticula of Manduca sexta and other
insects contains mainly N-acetyldopamine but the
hard, dark brown regions have mainly N-~alanyldopamine [128].
Many insect cuticulae contain fIrmly bound IJalanine. The incorporation of this amino acid into
the cuticula has been examined in detail for the
puparium of flies in which the haemolymph of larvae just prior to pupation contains large quantities of free ~-alanine or ~-alanyl dipeptides [138].
During sclerotization, the ~-alanine is incorporated into the cuticula. This process is disturbed
in some mutants of the fly Drosophila melanogaster and the beetle Tribolium castaneum [210]. The
Drosophila mutant "black" is unable to produce
~-alanine, and the mutant "ebony" cannot incorporate it into the epidermis; in both cases, the
cuticula of the adult animal is black instead of
brown. In isolated cuticulae, dopamine alone
produces a black colour, whereas N-~alanyldopamine gives the normal brown colour.
Thus, ~-alanine apparently blocks the amino
group of dopamine, inhibits melanin production,
and promotes the formation of sclerotizing structures from catecholamine [4, 118]. The localization and the chemical structure of ~-alanine
bonds in sclerotized cuticulae are not known
[4, 138]. It is also not clear whether, and in what
way, the differences in catechol spectra between
different insect species and between different
regions of the cuticula influence the process of
sclerotization and the properties of the sclerotized cuticula [157].
Compared with sclerotization of the insect cuticula, the hardening of the crustacean shell has
been relatively little investigated [23]. In the fiddler crab Uca pugilator, dopamine is converted to
N -acetyldopamine and incorporated into cuticular proteins of the post-moulting animal. In con-