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

Journal compilation © 2009 The Netherlands Entomological Society  99

DOI: 10.1111/j.1570-7458.2009.00831.x

BlackwellPublishingLtd

  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 Tr 

 

é

 

bicki

 

1,2

 

, Rob M. Harding 

 

1

 

& Kevin S. Powell

 

2

 

*

 

1

 

Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, GPO Box 2434,

Brisbane, 4001, Australia, and  2

 

Department 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

 

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 Tr

 

E

 

bicki, 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

of this leafhopper, the use of insecticides has been largely 

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 ordiet 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.

 

*

 

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

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100

 

Tr 

 

É 

 

bicki 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-cmdiameter 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

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 thesmaller 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 1m

 

potassium hydroxide. After filtration through a

0.2-

 

μ

 

m 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™ wasplaced over the chamber and 100 μ

 

l 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 

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.

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Screening anti-metabolites for leafhopper management 

 

101

 

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

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 theinclusion 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

 

Statistical analysis was performed using GenStat software

(10th Edition

 

©

 

2007, Lawes Agricultural Trust). A Kaplan–

Meier estimate and log-rank test were used to determinesurvivor 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,

 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 50L-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

1Source: Mitsuhashi, 1974.

Figure 2 Modified feeding chamber for

rearing Orosius orientalis on liquid diet

through a double layer of Parafilm M™.

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102

 

Tr 

 

É 

 

bicki et al

 

.

 

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-1did 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 Tr

 

E

 

bicki, 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 Tr

 

E

 

bicki, 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 haveimproved 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 andGNA 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 moreplant 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 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.

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Screening anti-metabolites for leafhopper management 

 

103

 

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 GNAbinds 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

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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