Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps.

Abstract

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.

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Journal of Insect Physiology 51 (2005) 1359–1365
Laccase-type phenoloxidase in salivary glands and watery saliva of the
green rice leafhopper, Nephotettix cincticeps
Makoto Hattoria,b,?, Hirosato Konishia, Yasumori Tamuraa,b, Kotaro Konnoa,
Kazushige Sogawac
aNational Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan
bInsect Gene Function Research Center, Tsukuba, Ibaraki 305-8634, Japan
cJapan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan
Received 24 June 2005; accepted 22 August 2005
Abstract
The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role
in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps,
its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants.
Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,20-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately
85kDa at pH 5. A band also appeared at a molecular mass of approximately 200kDa when the gels were treated with dopamine,
L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7.
The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase.
However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU).
Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200kDa).
Laccase activity was detected in a holidic sucrose diet that was fed on for 16h by two females, but only a trace of catechol oxidase activity
was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase
exhibited an optimum pH of 4.75–5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of
the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible
function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding
on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the
watery saliva.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: p-Diphenol:oxygen oxidoreductase; Salivary enzymes; Nephotettix cincticeps
1. Introduction
The green rice leafhopper, Nephotettix cincticeps (Uhler)
(Homoptera: Cicadellidae) is one of the most important
insect pests of rice in Japan. This species damages rice by
ingesting sap and also transmits virus and phytoplasma
diseases (O¯ya and Sato, 1981; Kawabe, 1985; Nakashima
and Hayashi, 1995). In the feeding process, leafhoppers
eject two different types of salivary secretions, namely,
coagulable and watery saliva (Sogawa, 1968). Coagulable
saliva rapidly forms the salivary sheath surrounding the
stylets that penetrate the host plant tissues, while watery
saliva remains in the liquid form after being ejected from
the stylets. It is considered that the saliva in aphids plays
important physiological roles in detoxifying toxic sub-
stances or in continuous ingestion of the sieve element sap
(Miles, 1999).
ARTICLE IN PRESS
www.elsevier.com/locate/jinsphys
0022-1910/$-see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2005.08.010
?Corresponding author. National Institute of Agrobiological Sciences,
Tsukuba, Ibaraki 305-8634, Japan. Tel.: +81298386085;
fax: +81298386028.
E-mail address: hatto@affrc.go.jp (M. Hattori).

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The saliva of leafhoppers contains several enzymes, as this
property has been reported in the case of other Hemiptera
species (cf. Miles, 1972; Sogawa, 1968, 1971). Based on
the reactions with various phenol compounds such as
L-3,4-dihydroxyphenylalanine (DOPA) and p-phenylene-
diamine, it has been demonstrated that the salivary glands
of N. cincticeps possess diphenoloxidase activity (Sogawa,
1968). However, it has not yet been proved whether the
enzyme involved is indeed a laccase (EC 1.10.3.2) in a strict
sense because the property of the enzyme has not been
examined by using laccase-specific substrates or inhibitors.
It also remained uncertain whether these activities are a
result of the functioning of a mixture of laccase and catechol
oxidase (EC 1.10.3.1) or of a single oxidase.
The phenoloxidase of N. cincticeps is inferred to be
involved in the process of stylet sheath formation and
undiffused out of the salivary sheath by an agar-gel test
(Sogawa, 1968). On the other hand, the salivary oxidases in
aphids are discharged in host plants and are considered to
detoxify defensive phytochemicals (Miles, 1964; Peng and
Miles, 1988; Urbanska et al., 1998). The electrical
penetration graph of N. cincticeps that was feeding on rice
phloem sap through a membrane recorded a distinct
waveform that was indicative of salivation (Hattori, 1997).
In the present study, the phenoloxidases in the salivary
glands of N. cincticeps were characterized and examined
further to determine whether they were secreted in watery
saliva for better understanding of their function in feeding
on the rice plants.
2. Materials and methods
2.1. Insects
The green rice leafhopper, N. cincticeps was obtained
from a stock colony successively reared on rice seedlings in
our institute at 25721C and 70%75% rh with a L16:D8
photoperiod. Unless otherwise stated, 4- to 7-day-old adult
females were used for the collection of salivary glands or
watery saliva.
2.2. Chemicals
DOPA, 2,20-azino-bis (3-ethylbenzthiazoline-6-sulfonic
acid (ABTS), syringaldazine, 3-methyl-2-benzothiazoli-
none hydrazone (MBTH), Kojic acid and catalase (from
bovine liver) were purchased from Sigma-Aldrich Co.,
St Louis, MO and hydroquinone, dopamine hydrochloride
catechol and 1-phenyl 2-thiourea (PTU) were obtained
from Wako Pure Chemical Industries Ltd., Osaka. N-
Hydroxyglycine was synthesized by the method of Jahngen
and Rossomando (1982).
2.3. Enzyme samples
After adult females were immobilized by placing them in a
freezer (?51C) for 15min, the salivary glands were dissected
out from the insects with tweezers. Salivation of the enzyme
was confirmed by measuring the enzyme activities of 5%
sucrose solutions that were exposed to insect probing and
sucking. For this purpose, glass cylinders (internal diameter,
28mm; height, 24mm) were used as feeding chambers. An
80-ml aliquot of 5% sucrose was dispensed between two
membranes of a stretched ParafilmsM; these membranes
were located at the upper end of the chamber. All
membranes were immersed in 70% ethanol over 1h and
subsequently dried prior to use. The bottom of the chamber
was enclosed with a piece of polyester net. Two adult females
were confined within each of the 10 chambers, which were
placed on a sheet of paper towel. ‘‘Unfed controls’’ were
aliquots of 5% sucrose solution that were dispensed in the
same manner but without any insects in the chambers. After
16h, 50-ml aliquots were withdrawn per chamber and
analyzed spectrophotometrically using a 96-well plate.
2.4. Electrophoretic analysis
Salivary glands were homogenized in Laemmli sample
buffer (Laemmli, 1970), but
omitted. After centrifugation at 15,000g for 10min, each
aliquot of the supernatant (equivalent to 15 pairs of
salivary glands) was subjected to different lanes of 10%
native polyacrylamide gel (PAGE) (NPU-10L, ATTO Co.)
under denaturing conditions using SDS. After electrophor-
esis at 41C, the gel was washed in 2.5% Triton X-100 for
15min at room temperature, rinsed twice with distilled
water and then immersed in individual buffer (pH 5 or 7)
for 10min. The gel was excised into individual lanes and
then activity staining was separately performed with
different substrates described in the next section.
Analytical isoelectric focusing (IEF) of the homogenates
was performed on a 5.0% acrylamide gel slab in the pH
range of 4.0–6.5 with a Multiphor electrophoresis system
(Pharmacia Biotech) used in accordance with the manu-
facturer’s instructions. The gels were stained with Coo-
massie brilliant blue (R250; Pharmacia) or with ABTS.
2-mercaptoethanol was
2.5. Visualization of enzyme activities within the gels
Laccase activity was detected by immersing the gels in
5mM ABTS, hydroquinone, or syringaldazine, typical
laccase substrates in 100mM acetate buffer (pH 5) after
electrophoresis. Catechol oxidase activity of the gels was
also assayed by the procedure of Nellaiappan and
Vinayagam (1986). This is based on the enzymatic
oxidation of diphenolic substrates such as DOPA, dopa-
mine and catechol into their quinone products and
subsequent condensation with MBTH to form a brown
product. The incubation mixture consisted of 4ml of
10mM of the substrates in either 100 mM sodium acetate
buffer at pH 5 or McIlvaine citrate–phosphate buffer at pH
7 added to 1ml of 0.3% MBTH in ethanol. Incubation was
carried out for 60min in the dark at 351C, except for a gel
that was treated with syringaldazine for 8h.
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2.6. Enzyme assay
The optimal pH range of salivary laccase was determined
colorimetrically by using ABTS as the substrate. Homo-
genates of two pairs of salivary glands in 10ml distilled
water were placed in each well of a 96-well plate. Forty
microliters of McIlvaine citrate–phosphate buffer with pH
ranging from 3.5 to 8.0 was added. The reaction was
initiated by the addition of 150ml of 2mM ABTS in
distilled water. The increase in absorbance at 420nm after
60min at 281C was associated with the oxidation of the
substrate and was measured on a microplate reader (Model
550; Biorad, USA).
In order to examine phenoloxidase activity in fed
sucrose diet, 150ml of ABTS in 100mM sodium ace-
tate buffer at pH 5 or 10mM dopamine in McIl-
vaine buffer at pH 7 was added to every 50ml of the
fed diet (two females for 16h) and unfed control diet in
the well.
2.7. Inhibition of enzyme activity
The effect of various inhibitors on salivary phenolox-
idase was evaluated by measuring the increase in the rate
of absorbance (min?1) at 420nm during the enzyme
reaction with ABTS or dopamine on a microplate reader
at 281C. The reaction mixture was composed of 40 ml
of salivary gland homogenates (two pairs), 150ml of
2mM ABTS in 100mM acetate buffer (pH 5) or of
10mM dopamine in McIlvaine buffer (pH 7) and 10ml of
various inhibitors in distilled water. N-Hydroxyglycine, a
specific inhibitor of laccase (Murao et al., 1992) and
other oxidase inhibitors with various concentrations were
added to the salivary gland homogenates 30min prior to
the addition of the substrate. In order to verify the
involvement of oxidation by peroxidase, catalase was
added to the homogenates at 333 and 1000units/ml to
scavenge H2O2.
2.8. Laccase activity of salivary glands in adult stage
Newly emerged adult females were transferred from
the rearing cage to another cage with rice seedlings
0–4h before the light was turned off. Everyday or every
2 days for a period of 12 days, five females were taken
from the latter cage and the laccase activity of sali-
vary glands in each insect was examined. In 0-day-old
females, the salivary glands were dissected within 1h after
eclosion.
2.9. Histochemical method
The salivary glands were fixed in 10% formalin in 0.8%
sodium chloride solution for 15min and incubated in 5mM
ABTS solution at 351C for 30min.
3. Results
3.1. Electrophoretic analysis by activity staining
When the salivary gland homogenates of N. cincticeps
were subjected to SDS-PAGE on a 10% gel under
nonreducing and no-heating conditions, followed by
activity staining with the laccase substrate ABTS at pH
5, a single band that was blue–green in color appeared
in the gel at a molecular mass of approximately 85kDa
(Fig. 1A). When the gel was incubated in ABTS and H2O2,
another band appeared at a molecular mass of approxi-
mately 15kDa as a result of the peroxidase activity (data
not shown).
The 85-kDa band was also visualized with hydroqui-
none, syringaldazine or dopamine at pH 5 and, DOPA and
catechol at pH 5 and pH 7. On the other hand, a red or
brown band appeared at a molecular mass of approxi-
mately 200kDa when the gels were treated with dopamine,
DOPA or catechol at pH 7 (Fig. 1A). No band appeared
on reaction with ABTS, hydroquinone and syringaldazine
at pH 7. IEF analysis with ABTS showed that laccase in
the salivary glands yielded a broad band with an isoelectric
point of approximately 4.8 (Fig. 1B).
3.2. Effect of inhibitors on two types of phenoloxidase
activity
We then proceeded toward the characterization of
salivary phenoloxidase reacting with ABTS at pH 5 or
with dopamine at pH 7. The salivary gland homogenates
were incubated in the presence of various types of
phenoloxidase inhibitors (Table 1). The phenoloxidase
activity detected with ABTS was completely inhibited by
ARTICLE IN PRESS
Fig. 1. (A) Nonreducing SDS-PAGE (10%) analysis of salivary gland
homogenates of N. cincticeps. 1,7: activity staining with ABTS; 2,8:
hydroquinone; 3,9: syringaldazine; 4,10: dopamine; 5,11: DOPA at pH 7;
6,12: catechol; 1–6: pH 5; 7–12: pH 7. (B) Isoelectric focusing (IEF) of the
salivary gland homogenates on a 5.0% acrylamide gel slab in the pH range
4.0–6.5. Left: Coomassie blue R-250 staining, right: activity staining with
ABTS at pH 5.
M. Hattori et al. / Journal of Insect Physiology 51 (2005) 1359–1365
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N-hydroxyglycine, a specific laccase inhibitor (Murao
et al., 1992), at the 0.1mM level, while dopamine-oxidizing
activity was not inhibited even at the 10 mM level. In
contrast, the ABTS-oxidizing activity was inhibited to a
lesser extent by PTU, while dopamine-oxidizing activity
was completely inhibited by PTU at the 1mM level.
Catalase reduced the ABTS-oxidizing activity by a meager
4.7% at a 333-U/ml concentration and did not perform the
dopamine-oxidizing activity even at 1000U/ml. Kojic acid,
a general polyphenol oxidase inhibitor inactivated both
oxidase activities at a similar level.
3.3. Optimum reaction pH of laccase
Salivary homogenates in distilled water were mixed with
McIlvaine buffer ranging from pH 3.5 to pH 6.5. As shown
in Fig. 2, the optimum reaction pH obtained by using
ABTS as the substrate was estimated to be 4.75–5. The pH-
activity curve is similar to that of cuticle laccase of Bombyx
mori and Schistocerca gregaria with an optimum pH of
4.75–5 (Yamazaki, 1972; Anderson, 1978), but is distinct
from polyphenol oxidase in the saliva of grain aphids,
which is most active at a pH of 8.2–9.4 (Urbanska et al.,
1998).
3.4. Salivary laccase activity at adult stage and
phenoloxidase activities in sucrose diet exposed to
leafhoppers
Laccase activity in the salivary glands was relatively low
on 0 day after eclosion; however, it was constantly high for
the period from 1 to 12 days after eclosion (Fig. 3). ABTS-
oxidizing activity of the sucrose diet exposed to feeding of
the leafhoppers was significantly higher than that of unfed
sucrose diet (Table 2). No significant dopamine-oxidizing
activity was recorded in the fed sucrose diet.
3.5. Histochemical localization of laccase in the salivary
glands and the salivary sheath materials
Laccase activity was detected in V-type cells of the
posterior lobe of salivary glands in female adults by using
ABTS as the substrate (Fig. 4). Catalase treatment did not
affect the staining intensity in these cells (not shown). The
V-type cells of the glands and the coagulated sheaths were
stained with DOPA (Sogawa, 1968).
4. Discussion
The phenol-oxidizing enzymes have been classified into
three groups based on their substrate specificity: mono-
phenoloxidase (EC.1.14.18.1),
(EC.1.10.3.1) and laccase (EC.1.10.3.2). The phenoloxidase
found in the salivary glands of N. cincticeps showed a
capacity to oxidize p-phenylenediamine and o-diphenols
catecholoxidase
ARTICLE IN PRESS
Table 1
Effect of various inhibitors on salivary phenoloxidase at different
concentrations
Inhibitor ConcentrationInhibition (%)
ABTS-oxidizing
activity
Dopamine-
oxidizing activity
p-Hydroxy
glycine
0.1mM100.0—
1.0mM
10.0mM
1.0mM
10.0mM
0.1mM
1.0mM
333U/ml
1000U/ml
100.0
100.0
39.4
87.9
2.8
50.0
4.7
10.0
—
0.0
45.7
100.0
52.0
100.0
—
0.0
Kojic acid
PTU
Catalase
The assay was performed with ABTS at pH 5.0 or dopamine at pH 7.0 and
the readings were taken at 420nm or 470nm, respectively. Activity was
expressed relative to those obtained in the absence of the inhibitors.
Fig. 2. Activity profile of salivary laccase; enzyme activity as a function of
pH in McIlvaine buffer. The activity was assayed with ABTS, and
readings were taken on a microplate reader at 420nm. Each point
represents Mean ? SE, n ¼ 10.
Fig. 3. Change in activity of laccase from the salivary glands of N.
cincticeps females in adult stage. In 0-day-old adults, the activity was
examined within 1h after eclosion. Mean ? SE, n ¼ 5.
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like catechol but not the monophenols such as tyrosine and
p-cresol (Sogawa, 1968). Sogawa (1968) has described that
this enzyme shows similar substrate specificity to a
phenoloxidase isolated from the larvae of the blowfly,
Calliphora erythrocephala (Karlson and Liebau, 1961), but
differs from the one that was prepared from the colleterial
gland of the cockroach Periplaneta americana (Whitehead
et al., 1960). Catechol oxidase (tyrosinase) and laccase have
been partially purified from the cuticle of larvae of C. vicina
(Barrett and Anderson, 1981) and catechol oxidase from
the colleterial gland of P. americana (Sugumaran and
Nellaiappan, 1990).
In order to clarify the property of the salivary
phenoloxidase of N. cincticeps and a possibility of a
mixture of different oxidases, the salivary gland homo-
genates were reacted with several phenoloxidase-specific
substrates after electrophoresis. It is well known that ABTS
is specifically oxidized by laccase in the absence of
hydrogen peroxide but not by catechol oxidase (Niku-
Paavola et al., 1990). Nonreducing SDS-PAGE of the
supernatant of salivary gland homogenates revealed a
single laccase-positive band at 85kDa by reacting with
ABTS at pH 5; this band was also reacted with
hydroquinone and syringaldazine, other diagnostic sub-
strates for laccase, and dopamine, DOPA and catechol at
pH 5. On the other hand, a band appeared at approxi-
mately 200kDa on intense staining with dopamine, DOPA
and catechol, substrates of catechol oxidase, at pH 7. Thus,
two different types of phenoloxidases were detected from
the salivary gland homogenates of N. cincticeps by using
various substrates at different pH values.
ABTS-oxidizing activity of the salivary gland homo-
genates was barely affected by catalase treatment, thereby
indicating little participation of peroxidase in oxidization.
Furthermore, the ABTS-oxidizing activity was completely
inhibited by N-hydroxyglycine at 0.1mM, while dopamine-
oxidizing activity was not inhibited even at 10mM. On the
other hand, the dopamine-oxidizing activity was distinctly
inhibited by PTU, while the ABTS-oxidizing activity was
inhibited to a lesser extent by PTU. N-Hydroxyglycine has
been identified as a potent and specific inhibitor of laccase
in Penicillium citrinum, but it did not inhibit other oxidases
such as polyphenol oxidase and ascorbate oxidase (Murao
et al., 1992); PTU are known as inhibitors of catechol
oxidase (Barrett and Anderson, 1981; Barrett, 1987).
Thus, these results indicate that the ABTS-oxidizing
enzyme with a molecular mass of 85kDa is laccase-type
phenoloxidase (EC 1.10.3.2, p-diphenol: oxygen oxidor-
eductase), and the dopamine oxidizing enzyme with a
molecular mass of 200kDa is catechol oxidase (EC
1.10.3.1, o-diphenol:oxygen oxidoreductase).
Thus far, insect laccase has been detected in the cuticles
of many species (Barrett, 1987; Thomas et al., 1989).
Recently, it was reported that a laccase gene was expressed
in the midgut, malpighian tubules and fat body as well as in
the epidermis of Manduca sexta (Dittmer et al., 2004). To
our knowledge, N. cincticeps laccase is the first laccase
found in the salivary glands, although catechol oxidase has
been universally identified in the salivary glands of
Hemiptera species (Miles, 1960; Miles, 1964; Peng and
Miles, 1988; Madhusudhan and Miles, 1998; Urbanska
et al., 1998).
Laccase activity in the salivary glands of N. cincticeps
was constantly maintained throughout the adult stage from
day 1 after eclosion—the period during which the
leafhoppers feed actively. One of the proposed functions
of the diphenoloxidase of N. cincticeps is the promotion of
rapid oxidative gelling of the stylet sheath by the quinone
tanning reaction because some polyphenol substance(s) has
been detected in the salivary glands (Sogawa, 1971, 1973).
This idea is conceivable because laccase is hypothesized to
be involved in the insect cuticle sclerotization (Yamazaki,
ARTICLE IN PRESS
Table 2
Two types of phenoloxidase activities in sucrose diet exposed to feeding of
N. cincticeps
Substrate
(2mM)
Absorbance at 420nm
After feedingUnfed control
ABTS, pH 50.11870.0090.06170.001
Po0.01
Absorbance at 470nm
After feedingUnfed control
Dopamine, pH
7
0.13670.0030.13570.002 ns
Eighty microliters of 5% sucrose was exposed to two adult females for
16h, and subsequently, 50ml was used for analysis with microplate reader
at 281C for 60min. Mean ? SE, n ¼ 10.
Fig. 4. Localization of laccase in a pair of salivary glands of a N.
cincticeps female. Blue–green colored parts are V cells in the posterior lobe
showing laccase activity on ABTS staining. The bar in the figure represents
100mm.
M. Hattori et al. / Journal of Insect Physiology 51 (2005) 1359–1365
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