DOI: 10.1111/j.1570-7458.2009.00831.

Blackwell Publishing Ltd

TECHNICAL NOTE
Anti-metabolic effects of Galanthus nivalis agglutinin
and wheat germ agglutinin on nymphal stages of
the common brown leafhopper using a novel artificial
diet system
Piotr Trébicki1,2, Rob M. Harding1 & Kevin S. Powell2*
1
Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434,
Brisbane, 4001, Australia, and 2Department of Primary Industries, Biosciences Research Division, Rutherglen,
Victoria, 3685, Australia

Accepted: 6 January 2009

Key words: lectin, GNA, WGA, cowpea trypsin inhibitor, bioassay, Orosius orientalis, vector,
oviposition, Homoptera, Cicadellidae

of this leafhopper, the use of insecticides has been largely

Introduction
The common brown leafhopper, Orosius orientalis
(Matsumura) (Homoptera: Cicadellidae), previously
described as Orosius argentatus (Evans), is an important
vector of several viruses and phytoplasmas worldwide.
In Australia, phytoplasmas vectored by O. orientalis cause
a range of economically important diseases, including
legume little leaf (Hutton & Grylls, 1956), tomato big bud
(Osmelak, 1986), lucerne witches broom (Helson, 1951),
potato purple top wilt (Harding & Teakle, 1985), and
Australian lucerne yellows (Pilkington et al., 2004).
Orosius orientalis also transmits Tobacco yellow dwarf
virus (TYDV; genus Mastrevirus, family Geminiviridae) to
beans, causing bean summer death disease (Ballantyne,
1968), and to tobacco, causing tobacco yellow dwarf
disease (Hill, 1937, 1941). TYDV has only been recorded
in Australia to date. Both diseases result in significant
production and quality losses (Ballantyne, 1968; Thomas,
1979; Moran & Rodoni, 1999). Although direct damage
caused by leafhopper feeding has been observed, it is
relatively minor compared to the losses resulting from
disease (P TrEbicki, unpubl.).
Control strategies for O. orientalis are primarily based
on the use of chemical insecticides (Paddick et al., 1971;
Paddick & French, 1972; Osmelak, 1986). However, due to
the paucity of information available on the life-cycle, popu-
lation dynamics, and disease transmission characteristics
*Correspondence: Kevin S. Powell, Department of Primary
Industries, Biosciences Research Division, RMB 1145, Chiltern Valley
Road, Rutherglen, Victoria, 3685, Australia.
E-mail: Kevin.powell@dpi.vic.gov.au
ineffective. An alternative approach to controlling sap-
sucking insect pests is the use of anti-metabolites, such as
plant lectins expressed in genetically modified plants
(Gatehouse et al., 1992; Peumans & Van Damme, 1995).
Using artificial diet bioassay systems, a wide range of plant
lectins, including Galanthus nivalis agglutinin (GNA) and
wheat germ agglutinin (WGA), have been shown to exhibit
anti-metabolic effects against many economically impor-
tant homopteran pests resulting in reduced survival and
delayed development (Habibi et al., 1993; Powell et al.,
1993; Gatehouse et al., 1995; Chen, 2008). Furthermore,
transgenic plants expressing lectins have shown enhanced
resistance towards many sap-sucking insects, including the
rice brown planthopper, Nilaparvata lugens (Stål) and
green leafhopper, Nephotettix spp. (Peferoen, 1997;
Gatehouse & Gatehouse, 1998; Jouanin et al., 1998). Based
on these studies, the use of anti-metabolites may also
potentially be an effective strategy to control O. orientalis.
A prerequisite to preliminary screening of potential
anti-metabolites in vitro is the development of a chemically
defined artificial diet for the target pest. The basic
nutritional requirements of O. orientalis are poorly under-
stood and, although artificial diets for several Cicadellidae
have been developed (Singh, 1977; Cohen, 2004), a diet or
diet feeding system has not been reported for O. orientalis.
In this study, we describe the development of the first arti-
ficial diet bioassay system for rearing O. orientalis in vitro
and a simple oviposition chamber for collection of newly
emerged first instars. In addition, we describe the use of
this diet bioassay system to assess the anti-metabolic effects
of two plant lectins and a protease inhibitor towards first
instars of the leafhopper.

© 2009 The Authors Entomologia Experimentalis et Applicata 131: 99–105, 2009

Journal compilation © 2009 The Netherlands Entomological Society 99
100 TrÉbicki et al.
Materials and methods
Insect culture
Stock colonies of O. orientalis were obtained from Charles
Darwin University, Darwin, Australia, and from herbaceous
vegetation surrounding commercial tobacco farms in
the Ovens Valley, north-east Victoria, Australia (36°37′S,
146°48′E). Leafhoppers were identified to species level
using morphological characteristics of the male genitalia
(Evans, 1966; Ghauri, 1966; Fletcher, 2000). Cultures were
maintained, on 30–40-day-old celery [Apium graveolens
L. (Apiaceae)] or bean [Phaseolus vulgaris L. (Fabaceae)]
plants grown under glasshouse conditions, for eight
generations before fecund female adults were transferred
to oviposition chambers and reared under controlled
environment conditions (25 ± 2 °C, L14:D10). The
oviposition chambers (Figure 1) were made by modifying
standard plastic Petri dishes (9 × 2.5 cm). Three access holes
on opposite sides of the dish base were made. A 3–4-cm
diameter opening was made in the lid and sealed with
glued mesh to prevent first-instar escape, facilitate air
circulation, and reduce condensation. One attached leaf of
the host plant was placed in the dish base with the stem
inserted into the access hole, covered with the lid and
sealed with an elastic band. The two remaining access holes
allowed introduction of fecund adult females and removal
of newly emerged first instars. To prevent insect escape
during oviposition or post-hatching, foam was wrapped
Figure 1 Oviposition chamber for Orosius orientalis on (A) host
plant with (B) rubber band closure, (C) fabric mesh ventilation
point, (D) Petri dish, (E) access points for nymph removal and
adult addition with foam plugs to prevent insect escape, and
(F) leaf attached to whole bean or celery host plant.
around the stem of the leaf and two foam stoppers were
placed into each access hole. Orosius orientalis oviposits on
the leaf blade, petiole, or stem (Helson, 1942), and using
this chamber design fecund females could freely choose
oviposition sites.
Chemicals and materials
Galanthus nivalis agglutinin and WGA were obtained from
Abacus Australian Laboratory Supplies, East Brisbane,
Australia. DL-homoserine was purchased from MP
Biomedicals Australasia, Seven Hills, Australia. Cowpea
trypsin inhibitor (CpTi) and all remaining dietary
components were obtained from Sigma-Aldrich, Castle
Hill, Australia. All chemicals utilized in artificial diet
preparation had the highest purity commercially available.
Artificial diet preparation
Two artificial diets were prepared for rearing nymphal
stages of O. orientalis, MED-1 as previously utilized for the
smaller brown leafhopper, Laodelphax striatellus Fallen
(Mitsuhashi, 1974), and a modification of MED-1
designated PT-07. Dietary modifications were made to
the amino acid and vitamin profile and concentration, and
by the addition of cholesteryl benzoate (Table 1). All
components were dissolved in sterile ultra pure water
using gentle heat (25 °C), and the pH was adjusted to 6.5
with 1 m potassium hydroxide. After filtration through a
0.2-μm Millipore disposable filter, diet solutions were
dispensed into 50-ml plastic containers as stock solutions,
and further dispensed into 1.5-ml Eppendorf tubes as
working solutions and stored at –20 °C prior to use.
Feeding trials
The effect of artificial diets, MED-1, and PT-07 on the
development and survival of O. orientalis was examined
using feeding chambers essentially as described by Powell
et al. (1993), but with a minor modification. The feeding
chamber was placed in a second, larger plastic Petri dish
(9 × 2.5 cm) containing wet filter paper to increase and
maintain constant humidity (Figure 2). Five newly
emerged first instars of O. orientalis were removed from
oviposition chambers with a fine wet paint brush and
placed in the feeding chamber (plastic Petri dish,
1 × 3.5 cm). A single layer of stretched Parafilm M™ was
placed over the chamber and 100 μl of diet was deposited
on the membrane. A second layer of Parafilm M™ was then
stretched over the artificial diet to form a feeding sachet.
Two controls (no diet and water only) were included in
all experiments and 10 replicates were used for each
treatment and control.
To examine the effect of anti-metabolites on the survival
of O. orientalis, GNA, WGA, and CpTi were separately
 Screening anti-metabolites for leafhopper management 101

Table 1 Composition (mg l−1) of artificial diets, PT-07 and MED-11, used for rearing Orosius orientalis; pH of both diets was adjusted with
KOH to 6.5

Ingredient PT-07 MED-1 Ingredient PT-07 MED-1

L-alanine 1 000 1 000 MgCl2·6H2O 2 000 2 000
γ-amino butyric acid 200 200 KH2PO4 5 000 5 000
L-arginine hydrochloride 3 000 4 000 CaCl2·2H2O 32 31.15
L-asparagine 4 000 3 000 CuCl2·2H2O 3 2.68
L-aspartic acid 1 000 1 000 FeCl3·6H2O 23 22.28
L-cysteine 500 500 MnCl2·4H2O 8 7.93
L-cystine hydrochloride 50 ZnCl2 5 3.96
L-glutamic acid 1 500 2 000
L-glutamine 6 000 6 000 Biotin 1 1
Glycine 400 200 Calcium pantothenate 50 50
L-histidine 1 500 2 000 Choline chloride 500 500
DL-homoserine 8 000 Folic acid 10 10
L-isoleucine 1 500 2 000 Inositol 500 500
L-leucine 1 500 2 000 Nicotinic acid 100 100
L-lysine hydrochloride 1 800 2 000 Pyridoxine hydrochloride 25 25
L-methionine 1 500 1 000 Riboflavin 25 50
L-phenylalanine 1 000 1 000 Thiamine hydrochloride 25 25
L-proline 1 000 1 000 Ascorbic acid 1 000
L-serine 1 000 1 000 Sodium ascorbate 1 000
L-threonine 1 500 2 000 Cholesteryl benzoate 25
L-tryptophane 1 000 1 000
L-tyrosine 200 200 Sucrose 50 000 50 000
L-valine 1 500 2 000
1
Source: Mitsuhashi, 1974.

incorporated into the PT-07 artificial diet at a concentra- Statistical analysis

tion of 0.1% (wt/vol). Five newly emerged first instars
of O. orientalis were used in each feeding chamber as
described above. ‘No diet’ and PT-07 diet without the
inclusion of a plant protein were included as controls. Ten
replicates were used for each treatment and the control.
In all feeding trials, leafhopper survival data were
recorded daily and diets were replaced on alternative days.
To avoid insect escape during the diet changing procedure,
insects were temporarily immobilized by placing the
feeding chamber at –20 °C for 90 s. To avoid fungal growth
on excreted honeydew, feeding chambers were replaced
weekly under aseptic conditions. Parafilm M™ and feeding
chambers were exposed to ultraviolet light for 20 min
prior to insect introduction. All trials were conducted in
controlled growth rooms (25 ± 2 °C, L14:D10).
Statistical analysis was performed using GenStat software
(10th Edition© 2007, Lawes Agricultural Trust). A Kaplan–
Meier estimate and log-rank test were used to determine
survivor distribution and to compare differences between
diet formulations and diets with anti-metabolic compounds.
Corrected mortality calculations (Abbott, 1925) were used
to compare the relative efficacy of treatments.
Results and discussion
Modifications were made to the MED-1 (Mitsuhashi,
1974) diet formulation resulting in the development of a
novel artificial diet PT-07. These modifications included
changing the proportions of amino acids (L-arginine
hydrochloride, L-asparagine, L-glutamic acid, glycine,

Figure 2 Modified feeding chamber for

rearing Orosius orientalis on liquid diet
through a double layer of Parafilm M™.
102 TrÉbicki et al.
Figure 3 The effect of two artificial diet formulations, MED-1
and PT-07, on the survival and development of first instars of
Orosius orientalis. Each data point represents the mean of 10
replicates, each of which contained five insects at the start of the
experiment.
L-isoleucine, L-leucine, L-lysine hydrochloride, L-
methionine, L-threonine, and L-valine), inorganic salts,
and including cholesteryl benzoate and ascorbic acid,
which resulted in enhanced survival and development of
O. orientalis.
PT-07 artificial diet was significantly more effective for
rearing O. orientalis than the MED-1 diet (Figure 3),
increasing development and survival. In the absence of
diet, using either no-diet or water-only controls, insects
survived for a maximum of 5 days. In contrast, the survival
of O. orientalis on the PT-07 diet was significantly
enhanced compared to the MED-1 diet and the controls
(log-rank test: P<0.001). Insects reared on the MED-1 diet
survived for a maximum of 14 days whereas the PT-07 diet
sustained insects for up to 46 days reaching adulthood at
day 42–45. When 100% mortality of leafhoppers was
reached on MED-1 diet, more than 80% of insects
remained alive on the PT-07 diet. Insects reared on MED-1
did not develop beyond the third instar (with a large
proportion not surviving ecdysis (data not presented),
whereas PT-07 supported leafhopper development to the
adult stage. On host plants leafhopper survival and develop-
ment varies depending on plant species as this insect is
polyphagous with up to 20 confirmed host plant species
(P TrEbicki, unpubl.). Under laboratory conditions, on
average it takes 25 days till nymphs reach adulthood when
reared on Malva parviflora L. (Helson, 1942) and up to
35 days on celery (P TrEbicki, unpubl.).
Liquid artificial diets for rearing homopteran pests
have been used for decades (Vanderzant, 1974) along with
a feeding sachet system for delivery which was first
developed for leafhoppers (Carter, 1927). Artificial diets,
which enable the rearing of Cicadellidae and Delphacidae
through successive generations (Mitsuhashi & Koyama,
1972; Mitsuhashi, 1974), have been developed but none
prior to this study was available for O. orientalis. Optimi-
zation of the concentration and proportion of amino acids
in a diet is an important factor for leafhopper development
and survival as they are known to have phagostimulatory
and phagoinhibitory properties (Sogawa, 1977). Some
leafhoppers also require a source of cholesterol for optimal
development (Lin & Hou, 1981). A number of amino
acids, including L-asparagine, were proven to act as a
sucking stimulant for N. lugens (Sogawa, 1972) and the
increased concentration of this amino acid may have
improved survival of O. orientalis on PT-07 diet.
First instars of O. orientalis were also exposed to the
PT-07 diet containing 0.1% (wt/vol) concentrations of either
GNA, WGA, or CpTi, with PT-07 diet only and no diet
used as a control. Using the PT-07 control only diet, some
insects were still alive after 43 days. Cowpea trypsin inhibitor
showed no significant effects on leafhopper survival or
development (Figure 4) with a corrected mortality of only
4%. In contrast, GNA and WGA both showed significant
anti-metabolic effects (log-rank test: P<0.001), with
nymphal survival reduced to 22 and 15 days, respectively.
Although the corrected mortality values using WGA and
GNA were relatively high at 37 and 35%, respectively and
significantly different to CpTi treatment (log-rank test:
P<0.001), they were not statistically different from one
another (P>0.1). These mortality levels, although lower
than those reported using brown planthopper (BPH)
N. lugens (up to 76%) (Powell et al., 1993), were nonethe-
less significant. The comparatively broad host plant range
of O. orientalis (Helson, 1942) may partially explain the
difference in mortality compared to N. lugens, which is
monophagus on rice. Other studies have shown that
more GNA binds to N. lugens gut tissue compared to
corresponding tissue of the rice green leafhopper (GLH),
Nephotettix spp., despite the fact that GLH ingested more
plant sap from GNA-transformed rice plants (Foissac
et al., 2000).
Previous studies, using either an in vitro artificial diet
bioassay system for screening anti-metabolites or by
expressing proteins in transgenic plants, have shown that a
number of plant-derived compounds affect the development
of a range of Homoptera. GNA and WGA, for example,
have shown significant anti-metabolic effects towards

Figure 4 The effect of Galanthus nivalis agglutinin (GNA), wheat
germ agglutinin (WGA), and cowpea trypsin inhibitor (CpTi)
when incorporated at 0.1% (wt/vol) in artificial diet PT-07 on the
survival of first-instar nymphs of Orosius orientalis. Each data
point represents the mean of 10 replicates, each of which
contained five insects at the start of the experiment.
the rice GLH (Nephotettix cinciteps Uhler) and rice BPH
(N. lugens) (Powell et al., 1993, 1995a,b; Foissac et al.,
2000). Immunolabelling studies have shown that GNA
binds to cell carbohydrate moieties in the gut of BPH,
causing a granular appearance of the mid-gut epithelial
cells, as evidenced by disruption of the microvilli (Powell
et al., 1998). GNA has also been shown to be an effective
anti-metabolic agent for several aphid species (Sauvion
et al., 1996), causing delayed development and reduced
survival.
In this study, CpTi showed no significant effects on leaf-
hopper survival or development. Previous studies have
shown that, although CpTi incorporated into artificial
diets or expressed in transgenic plants reduce survival in
some lepidopteran (Hilder et al., 1987; Bell et al., 2001),
coleopteran (Graham et al., 1997), and orthopteran
(Boulter et al., 1989) species, this protein was not considered
effective enough to be regarded as a viable control agent for
Homoptera (Boulter et al., 1989). Similar observations were
obtained from N. lugens and N. cinciteps fed on artificial
diets containing CpTi (Powell et al., 1993).
In our study, two plant lectins, GNA and WGA, have
been identified as potential control agents for O. orientalis.
However, further in vitro and in planta studies are required
Screening anti-metabolites for leafhopper management 103
to determine the effectiveness of this approach in trans-
genic crops. The mechanism of action of these lectins
towards O. orientalis also requires further investigation as
this could impact on its effectiveness as a vector. Both GNA
and WGA have been shown to have an antifeedant effect
against planthoppers (Powell et al., 1995b), resulting in
increased probing activity (Powell & Gatehouse, 1996) and
both lectins also bind to insect mid-gut epithelial cells
(Eisemann et al., 1994; Powell et al., 1998) and this is
dependant on the binding site affinity with GNA and WGA
binding to d-mannose and N-acetyl glucosamine sites,
respectively (Sharon & Lis, 1989).
In previous studies, several crop species including
tobacco have been genetically modified to express GNA
and this approach has led to reduced survival of aphids and
planthoppers (Hilder et al., 1995; Rao et al., 1998; Stoger
et al., 1999; Chen, 2008). Because current chemical control
agents for O. orientalis have little effect in reducing the
incidence of this leafhopper and the diseases it transmits,
expressing lectins with different modes of action in trans-
genic plants through gene pyramiding (Burrows et al., 1999)
may be an alternative strategy to provide more effective
control and to combat the development of potential
resistance-breaking genotypes. However, selection of an
appropriate promoter gene could also influence the
epidemiology of disease transmission. A comparison
between constitutive promoters or phloem specific
promoters (Wang et al., 2005) would be advisable for
O. orientalis as TYDV is phloem restricted (Needham
et al., 1998).
Acknowledgements
This research was funded by Horticulture Australia Limited
and the Tobacco Research and Development Corporation
with in-kind support from the Department of Primary
Industries (DPI), Victoria, Australia. The assistance of
Brendan Rodoni (DPI Knoxfield), Gary Baxter (DPI
Ovens), and Lucy Tran-Nguyen (Charles Darwin University,
Australia) is gratefully acknowledged.
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