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ACARICIDE RESISTANCE IN NORWEGIAN POPULATIONSOF THE TWO-SPOTTED SPIDER MITE (Tetranychus urticae Koch) (Acari: Tetranychidae)

CASSIUS GARWO VARNEY FAHNBULLEH

i

D E C L A R A T I O N

This thesis has not previously been accepted for any degree and is not been currently

submitted in candidature of any other degree. This work is the result of my own

investigation. All other sources of information are acknowledged and a reference list

appended.

Cassius G. V. Fahnbulleh

Sgd:…………………….

Date:…………………….

ii

D E D I C A T I O N

This piece of work is dedicated to my parents (Mr. William Siaka Fahnbulleh and Ms.

Maima Gbeni Fahnbulleh), (though deceased) for their care over me. Parents, because of

your farsightedness and through God – I am whom I am. It is also dedicated to my two

children (Musukula D. Fahnbulleh and William S. S. Fahnbulleh) who toiled alongside

me and endured hardship throughout those dreadful years of exile in Sierra Leone. Their

patience, understanding and continued love are highly valued – may God illuminate the

paths on which they tread.

iii

A C K N O W L E D G E M E N T S

In my quest approximately a year ago for a suitable topic to write a thesis, I was fortunate

to have had one from Bioforsk in the area of entomology – though challenging. But

during the course of the work, I had access to many valuable, enthusiastic and inspiring

literatures and discussions on the topic from several quarters at Bioforsk lab that

broadened my horizon; for this I am grateful.

The lab work was interesting, at some point in time apprehensive and stressful but mostly

cheerful and absorbing because of the new knowledge. Several people contributed to this

work, knowingly or unknowingly in diverse ways. They are too many to allude to by

name but to mention a few: I wish to extend my sincere gratitude to my immediate

supervisor, PhD. Einar G. M. Nordhus, for his excellent guidance and immense and

valuable contributions in both the laboratory work when I have felt confused in the use of

strange gadgets, and the writing processes when the construction of my thoughts in words

became an impediment. I am particularly indebted to my main supervisor, Prof. Arild

Andersen, who readily consented to work along with PhD. Nordhus to ensure that the

work is carried out at the Bioforsk lab – to you I am thankful. Not forgetting Dr. May-

Guri Sæthre, who besides suggesting the topic for this work, made it possible for me to

meet Dr. Nina S. Johansen through whom I became acquainted with my supervisors, my

gratitude. I must equally acknowledge the willingness of Toril Sagen of the aforesaid lab

who was always ready to assist in anyway possible for this work to be successful. In

addition, a big thank you goes to all other staff members attached to Bioforsk for their

iv

helpfulness and good nature towards me during my association with them, this will

always linger in my memory.

Last, but of course not the least, my study at UMB could not have been possible without

the humanitarian gesture of the people and government of Norway, who through the UN

resettlement programme, wholeheartedly granted me and my family asylum to recreate

our devastated lives after languishing in refugee camps and slums in Sierra Leone for 13

successive years where we nearly met our demise. For this, I am sincerely grateful.

Above all, I thank Almighty God for preserving my life.

Ås, 2007

Cassius G. V. Fahnbulleh

v

C E R T I F I C A T I O N

This is to certify that this thesis work was carried out by Cassius G. V. Fahnbulleh in the

Bioforsk laboratory, The Norwegian Institute for Agricultural and Environmental

Science, Norwegian University of Life Sciences (UMB), Ås.

Sgd: ……………………….. Approved: ………………....

PhD. Einar Nordhus Prof. Arild Andersen

Thesis Supervisor Main Supervisor

Date:………………. Date:………………..

vi

ABSTRACT

Farmers and greenhouse crop growers are faced with numerous polyphagus and

phytophagus pest species, among which is the two-spotted spider mite, Tetranychus

urticae Koch (Acari: Tetranychidae). This species has become a deleterious economic

pest in many cultivated crops, and its control has posed serious threats to farmers because

of its rapid growth rate and speedy adaptation to numerous acaricides. This study is

therefore aimed at identifying and characterizing the resistance in two Norwegian

populations of the two-spotted spider mite, T. urticae compared to a susceptible strain

GSS.

Standard bioassay procedures were used to determine the resistance in T. urticae by spray

application of different rates of acaricides against the different strains of spider mite

larvae. Mortality was scored after 72 hrs. through data collection, analyzed and results

expressed in Abbott’s corrected percent mortality and the LC50 values and slopes were

estimated by probit analysis, and resistance factor (RF) calculated.

The resistance factor (RF) for NOR4 at LC50 for abamectin was twice more than the

susceptible strain (GSS). NOR4 showed low levels of resistance to hexythiazox,

clofentezin and fenazaquin respectively compared with GSS; it also showed no level of

resistance to spirodiclofen and fenpyroximate. Unlike NOR4, NOR5 showed moderate

level of resistance to abamectin but with low level of resistance to clofentezine and

hexythiazox compared to the GSS strain. But it showed no resistance to spirodiclofen

fenpyroximate and fenazaquin. This implies that they exhibited certain level of

susceptibility to these drugs.

vii

TABLE OF CONTENTS

PAGE

Declaration i

Dedication ii

Acknowledgements iii-iv

Certification v

Abstract vi

Table of Contents vii-viii

Abbreviations ix-x

1. INTRODUCTION 1

1.1 General Introduction to Two-spotted Spider Mite, Tetranychus urticae (Koch) 1-3

1.2 Resistance 3-6

1.2.1 Resistance Mechanisms 7

1.2.1.1 Metabolic Mechanisms / Detoxification Mechanisms 7-10

1.2.1.2 Target Site Insensitivity / Resistance 10-11

1.2.2 Resistance in Norway 12

2. MATERIALS AND MATHODS 12

2.1 Strains 12

2.2 Miticides 12-13

2.3 Bioassay Procedure 13-14

2.4 Statistical Calculations 14

3. RESULTS 15-26

4. DISCUSSIONS 27-29

viii

5. CONCLUSIONS 29-30

6. REFERENCES 31--44

7. APPENDIX 45-48

x

ABBREVIATIONS

AChE Acetylcholine esterase

CO Company o C Degree Celsius

Cons Concentrations

DDT dichlorodiphenyltrichloroethane

DF

EC Emulsion concentrate

GABA γ - Aminobutyric acid

GSS German susceptible strain

GST Glutathione S-transferase

g / ldH20 gram / litre distilled water

hrs hours

IPM Integrated Pest Management

IRAC Insecticide Resistance Action Committee

kdr knockdown resistance

LC50 Median lethal concentration of a pesticide expected to kill 50% of a test

organism

LC90 Lethal concentration of a pesticide expected to kill 90% of a test organism

L: D Light to darkness

LTD Limited

METI Mitochondrial electron transport inhibitor

MFO Cytochrome P450 –dependent monooxygenase (Mixed function oxidase)

NADH dehydrogenase

NIAER Norwegian Institute for Agricultural and Environmental Research

NOR4 Norwegian strain no. 4

NOR5 Norwegian strain no. 5

OP Organophosphate

RAC Resistance Action Committee

RF Resistance factor

xi

RR Recommended rate

RH Relative humidity

SC Suspension concentrate

UK United Kingdom

WHO World Health Organization

1. INTRODUCTION

1.1 Two-spotted Spider Mite, Tetranychus urticae (Koch)

The two- spotted spider mite or glasshouse red spider mite, Tetranychus urticae Koch, is

widely known as a serious pest of economic importance in agriculture. The name ‘spider

mite’ emanates from the silk webbing made by mites and not because they appear like

small spider (Zhang, 2003). They are not the only web-spinning pests to feed on plants,

but they are the most common. T. urticae is a phytophagus mite and causes significant

yield losses in many horticultural, ornamental and agronomic crops globally (Jeppson et

al., 1975; Van de Vrie et al., 1972).

Figure 1. A) A rose plant and B) Two spotted spider mite on a rose plant

The two-spotted spider mite belongs to the class Arachnida (arachnids) which contains

four pairs of legs in contrast to that of insects. It has five life stages i.e. egg, larva,

protonymph, and deutonymph (or nymphs), and adult. At emergence from the egg, the

larva is colourless and has three pairs of legs, it also has red eyes. After eating, the colour

may change to light green, brownish yellow or dark green. At this developmental stage,

two dark spots appear in the middle of the body. Upon eating enough food, the larva goes

through a rest period until it develops into protonymph; it is at this time that the four pair

of legs appears. At this stage, the legs are little bigger, and the colour of the mite differs

from light to deep green. The two spots enlarge and appear clearer than on the larva.

After further feeding, the protonymph also goes into dormancy and then develops into a

A. B.

2

deutonymph which appears bigger but with similar coloration to the protonymph. At this

stage, differences can be seen between the male and the female. The female is slightly

bigger and rounder in shape than the male which is oblong in shape. It is from the

deutonymph that the adult mite develops once the nymph has had both a feeding and a

dormancy period.

Due to its rapid proliferation and very short life cycle coupled with favourable climatic

conditions, many generation of T. urticae can be completed in a growing season

(Crooker, 1985; Helle & Sabelis, 1985a). Females can, under favorable conditions,

oviposit approximately 30-50 eggs in 24 hours on the surface of plant structures, often

hidden in fissures. The eggs take 1-2 days to hatch with an approximate temperature of

22 (±1) 0C (Zhang, 2003). Details included in Table 1. (Herbert 1981) demonstrate the

growth periods at 210 C for individual larval and nymphal stages of both sexes. This

shows that female takes longer to develop than males. Mature males will remain near the

female nymph until the mature female emerges, and copulation takes place almost

immediately after emergence.

Table 1.Development time in days for Tetranychus urticae Koch at 210 C (Herbert, 1981)

The development rates of mites are dependent upon temperature, species, host plant,

humidity and other factors. From egg to adult may vary from 5-10 days based on the

Active Quiescent Total

Larva Male 1.5 1.3 2.8 Female 1.5 1.2 2.7 Protonymph

Male 1.0 1.3 2.3

Female 1.3 1.2 2.4

Deutonymph

Male 1.0 1.4 2.5

Female 1.5 1.4 2.9

3

aforesaid condition(s). Carey & Bradley (1982) discovered the growth stages in T. urticae

from egg to adult to take only 6.2 days at 29.40 C; while the optimum temperatures for

development is found to be 30-320 C (Crooker, 1985; Zhang, 2003). This implies that the

two-spotted spider mite can complete a generation as little as 5 days under the most

favorable conditions. Mites particularly tend to increase during rainless periods. The two-

spotted spider mite multiplies rapidly during period of low humidity and any conditions

when growers reduce misting or spraying indoor (Zhang, 2003), and during periods with

low precipitation outdoors.

Both immature and mature T. urticae suck up leaf fluid and principal photosynthetic

pigment (chlorophyll) necessary for photosynthesis. This feeding habit, if severe, may

cause browning of the leaves, reduction in photosynthesis, and eventually leaf death.

Fruit set can be reduced the following year, and fruit sizes may become smaller (Polk,

1994; Barkette, 1994). In some crops, a small amount of injury can lead to high economic

loss e.g. in cut flowers (Croft & Van de Bann, 1988). The main problem with the species

in Norway is in flower production in greenhouses and strawberry production under field

condition.

1.2 Resistance

The WHO (World Health Organization) defined resistance as the inherited ability of a

strain of some organisms to survive doses of toxicant which would prove harmful to a

greater number of the individuals in a normal community of the same strain (Brown &

Pal, 1971). Yet another definition propounded by Sawicki (1987) states that: Resistance

represents a genetic modification in reaction to assortment by poisons that may hinder

control in the field. Generally, pests are known to develop resistance to chemical(s) due

to uncontrolled use and weak effect from common pesticides which are not actually

potent against them; and this resistance, in part, emanates from their genetic makeup

(Zhang, 2003).

4

The development of pesticide resistance takes a multi facet dimensions. For this reason,

worldwide cases of resistance combined with scientific and public pressures led the

pesticide industry to form various ‘resistance action committees - RACs’ including one

for insecticide (i.e. Insecticide Resistance Action Committee - IRAC). This group, not

been in total agreement with the above definition of resistance, came out with criteria to

be considered as additional reasons for resistance development (Tomlin, 1997). They

include the following: that the chemical product has a record of not been potent against

the particular pest in question; that the product lack of success is not due to poor storage,

adulteration, or application, nor caused by abnormal climatic or environmental

conditions; that the recommended dosage failure to suppress pest populations below the

level of economic threshold, and that failure to control is due to heritable change in the

susceptibility of pest population of the product. Based on the above criteria, IRAC

pointed out that the term ‘resistance’ should be used only when field failure occurs and

this situation is confirmed (Mota-Sanchez et al., 2002). Resistance can also be considered

as a form of self-defense, because pest subjection to the many environmental stresses or

dangers such as: humidity, temperature, radiation, predation/parasitism, diseases, and

pollutants in the form of pesticides or plants allelochemicals inhibit their activities for

survival. It is the apparent expression of pests’ natural response to the above stresses

through resistance mechanisms that is regarded as self-defense (Koehn & Bayne, 1989;

Scott, 1995).

Resistance can be classified as:

a) Cross-resistance i.e. the resistance to two or more types of pesticides because they

have identical or very similar mode of action. Ex. Organophosphate (OP) and carbamate

pesticides intoxicate by similar moods of action, and resistance to one usually results in

resistance to the other;

b) Multi-resistance i.e. resistance to two or more types of pesticides because of the

coexistence of two or more different resistance mechanisms. For instance, a resistant

insect may have both metabolic resistance to OPs and target-site resistance to pyrethroids

(Scott, 1995).

5

Other form of resistance development in pests include: behavioral resistance (i.e. to avoid

toxic dose of the chemical that would otherwise prove deadly) (Roush & Mckenzie,

1991).

It has been found that T. urticae has the potential to speedily develop resistance against a

large number of acaricides (Cranham & Helle, 1985; Knowles, 1997; Stumpf & Nauen,

2001). Furthermore, their high prolific rate, their rapid growth period, their polyphagus

feeding habit, coupled with their extremely dispersal behaviour (i.e. omnipresent-ness) all

help to boost resistance development in the species (Croft & Van de Bann, 1988). The

persistent exposure of T. urticae to diverse pesticides in order to contain them below

economic threshold has resulted in resistant populations found in more than 40 countries

in both greenhouses and field conditions (Georghiou & Lagunes-Tejeda, 1991), and

resistance to at least 85 different compounds has been published

(http://www.pesticideresistance.org).

There are many different classes of miticides commercially available. Some of the most

used in Norway are:

(a) METI (mitochondrial electron transport inhibitors) acaricides that can prevent and

allow mitochondrial respiration (Hollingworth & Ahammadsahib, 1995; Wood et al.,

1996). They are known to be effective against all development phases of tetranychid,

tarsonid and eriophyd mites (Tomlin, 2003). They function by hampering complex I

(NADH: ubiquinone oxidoreductase) of the mitochondrial respiratory chain (Tomlin,

2003; Hollingworth & Ahammadsahib, 1995). Resistance was first reported in

Tetranychus kanzawa (Kishida) which was collected from tea fields in Japan in 1994

(Ozawa, 1994). Resistance of T. urticae to METI acaricides has been found in England

(Devine et al., 2001; Stumpf & Nauen, 2001; Sato et al., 2004), Australia (Herron &

Rophail, 1998) and Belgium (Bylemans & Meurrens, 1997). This resistance is said to be

inborn as an incomplete dominant trait and has been associated with over-production of

detoxification enzymes like esterases (Devine et al., 2001; Stumpf & Nauen, 2001; Sato

et al., 2004).

6

(b) Mite growth inhibitors are another group of insecticides. They have little or no effect

on mature females, but can cause them to lay fewer viable eggs (Chapman & Marris,

1986). The effect of these acaricides is mostly on juveniles (nymphs), by preventing

moulting. Resistance to mite growth inhibitors has been detected in the European red

mite and the two-spotted spider mite from different countries (Nauen et al., 2001). For

example, hexythiazox is thought to disrupt chitin fusion (Flexner et al., 1995; Dekeyser &

Downer, 1994); it has been reported to be selective, and it showed no effect on adult

carnivorous mites making it well suited in integrated pest management (IPM) (Hoy &

Ouyang, 1989). Another mite growth inhibitor, clofentezine, resistance was detected for

the first time in T. uraicae in 1987 on Australian roses (Edge et al., 1987), and in 1998 on

apple (Herron & Rophail, 1998). Cross resistance between the ovo-lavicides is well

known (Stumpf & Nauen, 2001). Such cross-resistance between hexythiazox and

clofentezine is likely not the result of metabolic traits, but an alteration in the target site is

proposed to be the possible cause resulting in the high levels of resistance observed in

these populations. However, the study of target site resistance as regards to hexythiazox

and clofentezine is presently not possible because the site of action is not known (Stumpf,

2001).

(c) Another chemical group, chloride channel activators, has also been used a lot against

mites. These chemicals are antagonists of the γ -Aminobutyric acid (GABA) receptor and

increase transfer of chloride-ions which will lead to mite paralysis and death (Fritz et al.,

1979; Matsumura et al., 1987). Resistance in T. urticae to abamectin is known from

California, Florida, Netherlands (Campos et al., 1996), and Washington (Beers et al.,

1998).

(d) Spirodiclofen is a recently developed acaricide with a good potency against mites.

The compound is particularly active against the developmental stages of the mite and not

so much against the adults (Nauen et al., 2000). The mood of action is not known and no

report of resistance has been published.

7

1.2.1 Resistance Mechanisms

Although there are many different chemical pesticides and different species of insects and

mite pests, the number of processes known to cause resistance to widely-used chemical

pesticides is scarce (Scott, 1995). In light of the above, two known resistance processes

are discussed. They are metabolic resistance (detoxification mechanisms) and target site

insensitivity /resistance.

1.2.1.1 Metabolic Resistance / Detoxification Mechanisms

Metabolic resistance is the potential of pests to expel poisonous pesticides from their

body through chemically driven deterioration. Three enzyme families have been found to

be involved in this resistance mechanism: Nonspecific esterases, cytochrome P450 –

dependent monooxygenases and glutathione- S –transferases (GSTs) (Brogdon &

McAllister, 1998).

Cytochrome P450 – dependent monooxygenases

The cytochrome P450 - dependent monooxygenases belong to a vast super family of

water-repelling, hemecontaining enzymes involved in the detoxification of many

identical internally originated elements such as juvenile hormones, ecdysteroids and

pheromones, and externally originated substances such as plant allelochemicals,

insecticides and promutagenes (Eldefrawi et al., 1960; Nordhus, 2005). As for its

association with acaricide resistance, the process of fluvalinate resistance in the Varroa

mite is a good example (Hillesheim et al., 1996). Cytochrome P450 oxidizes biochemical

change in insecticides through O-, S-, and N- alkyl hydroxylation, aliphatic hydroxylation

and pixilation, aromatic hydroxylation, ester oxidation and nitrogen and thioester

oxidation (Wilkinson, 1976; Maitra et al., 1996; Brogdon & McAllister, 1998).

The first account on P450 monooxygenase resistance became apparent in the 1960’s when

it was revealed that the resistance was eliminated by the P450 suppressor desamex

(Nordhus, 2005). From that period onward, P450 monooxygenase mediated resistance has

8

been known to be in a variety of different insects, and can be seen as the most repeated

type of metabolic resistance in entomology (Scott, 1991; Nordhus, 2005). Both increased-

adjustment and amino acid replacements have been associated with increased metabolism

of insecticides by different P450. For example, a monooxygenase CYP6D1 in housefly, M.

domestica, has been associated with pyrethroid resistance through increased production

(Kasai & Scott, 2000; Nordhus, 2005). Furthermore, resistance against DDT has been

associated with amino acid replacement in P450 CYP6G1 in Drosophila strains (Berge et

al., 1998; Nordhus, 2005). Due to the high number of P450 monooxygenases in insects and

the frequent-overlapping substrate restricted to an individual species of this enzymes

group, it has been difficult to discover a good biochemical structure and molecular way

of showing the monooxygenase-mediated resistance. Some in vivo inhibition methods

have been made to study the involvement of P450 in resistance. For example, synergists

like piperonyl butoxide are used to suppress P450 activity in bioassays to elucidate

resistance involvement (Nordhus, 2005). The over-expression of P450 monooxygenases

has also been associated with resistance to a variety of categories of insecticides inclusive

of neonicotinoids, OP and growth regulators (Daborn et al., 2001; Le Goff et al., 2003;

Nordhus, 2005). Cytochrome P450-dependent monooxygenases, which is also considered

as mixed function oxidases (MFO), are one of the most vital metabolic systems (phase I)

in insects and supposedly in mites as well. The MFO-based mictrotiter plate assay using

P-nitroanisole as a substance on which an enzyme acts in biochemical reactions

developed by Rose et al. (1995), was altered for exact characterization of cytochrome

P450 - dependent monooxygenase action in spider mite microsomes. But no specific

resistance mechanisms have yet been found to be associated with it.

Glutathione- S - transferases (GSTs)

Glutathione –S – transferase is another enzymes class that has been found to be involved

in metabolic resistance (Scott, 1995). It is a diverse class of enzymes that take major part

in the detoxification of both internal and foreign substances including insecticides

(Salinas & Wong, 1999; Nordhus, 2005). They speed up the degradation of pesticides or

their metabolism (Stumpf, 2001) so as to increase water solubility and later the

9

elimination of lipophilic substances (Motoyama & Deuterman, 1975; Stumpf, 2001).

They can convert the poisonous substances of pyrethroid, organophosphate (OP), DDT,

cyclodiene and carbamate insecticides into harmless substances. An increased GST

production has been associated with resistance to all major classes of insecticides

(Prapanthadara et al., 1995; Vontas et al., 2001; Hemingway et al., 2004; Nordhus, 2005),

but the process involved in this elevated enzyme production is not well known (Anayati

et al., 2005; Nordhus, 2005). Enzymatic detoxification has the potential to confer cross-

resistance to toxins independent of their target site (Stumpf, 2001). Despite the fact that

much is known about insect GSTs and their role in the biochemical changes (metabolism)

of insecticides, little information is available concerning the enzyme in spider mite

(Stumpf, 2001) therefore, resistance mechanisms linked to GST in mites are difficult to

elucidate.

Nonspecific Esterases

Esterases are a large group of enzymes which break down a large number of different

substrates. All esterase enzymes are able to split ester bonds in the presence of water, and

because most insecticides, particularly organophosphates (OPs) and carbamates have

ester bonds, they are prone to esterase degradation. The resistance mechanisms in most

cases are a result of increased levels of esterases production (Fournier et al., 1987; Field

et al., 1988; Carlini et al., 1991; Kettermen et al., 1992; Chen & Sun, 1994; Scott, 1995).

This increased esterase production process is known in mosquitoes, houseflies, mites and

whiteflies respectively (Ahmed & Wilkins, 2002; Hemingway & Karunaratne, 1998;

Nordhus, 2005). In Myzus persicae Sulz and Culex mosquitoes, the esterases genes which

give rise to resistance are highly amplified and about 250 copies of the similar gene may

be seen in a single individual (Mouchès et al., 1986; Poiriè et al., 1992). The more the

esterase genes are ampilifed, the higher the level of resistance provided (Field et al.,

1988; Poiriè et al., 1992). Two point mutations in the region of binding and chemical

reaction of substrate of the E3 malathion carboxyl esterase in a strain of sheep blow fly,

Lucilia cuprina, cause resistance to a range of OPs (Campbell et al., 1998; Nordhus,

2005). Almost identical mutations have been noticed in several malathion resistant

10

Anopheles species (Herath et al., 1987; Hemingway, 1982; Nordhus, 2005) and in a

housefly strain (Claudianose et al., 1999; Nordhus, 2005). A considerable amount of

evidence shows that esterases are connected with acaricide resistance in many spider mite

strains. For instance, Kim and Lee (1990) obtained resistant strains of T. urticae by

sequential assortment with the acaricides: carbophenothion, ethion, dicofol, cyxehation

and bifenthrin, and separated easterase isoenzyme by polyacrylamide gel electrophoresis.

By doing so, they were able to detect different isoenzymes in the resistance and

susceptible strains. Weyda et al. (1984) suggested that there were different esterase

arrangements and particular size numbers in polyacrylamide gel electrophoresis between

strains resistance and susceptible to thiometon. The difference indicated that esterases are

associated to the resistance mechanisms of the tested acaricides. Capua et at. (1990) and

Sundukov et al. (1989) supported their finding based on their own studies.

1.2.1.2 Target site insensitivity / resistance

Target site resistance involves the modification of the structures that are the target sites

within the insect or mite which leads to a change in susceptibility, so that the target is less

reactive to pesticide action (Stumpf, 2001). At least three different target site resistance

mechanisms have been found: γ - aminobutyric acid (GABA), Acetylcholine esterase

(AchE) and Sodium channel (knockdown resistance, kdr). All of these are well known

targets of insecticides, and resistance alleles of each have been found (Scott, 1995).

A modification in the main anatomy of acetylcholinesterase (AChE) can minimize the

level of suppression by OPs and carbamates, and causes resistance in insects and other

arthropod species. An unresponsive AChE resulting in OP resistance is found in many

different insect species and has also been discovered in T. urticae strains from Germany

(Voss & Matsumura, 1964; Smissaert et al., 1970), New Zealand (Ballantyne & Harrison,

1967), and in a few other tetranychid pest species, including the carmine spider mite (T.

cinnabarinus) from Israel (Zahavi & Tahori, 1970), the kanzawa spider mite, T. kanzawa,

from Japan (Kuwahara, 1982) and Caloglyphus berlesei (Blank, 1979). Diverse

molecular studies with pests revealed that minimized responsiveness of AChE is

11

attributed to one or more point - mutations in the gene resulting in modification of

structures in the enzyme (Fournier et al., 1992; Stumpf, 2001 ); it has been revealed that

successions with several mutations can bring about higher levels of resistance (Stumpf,

2001). Two point mutations in the voltage - gated sodium channel (kdr) have been found

to increase the resistance to pyrethroids and DDT. The first mutation termed kdr produces

a 10 to 20 fold resistance, while in combination with the second mutation (super-kdr) the

resistance level can increase more than 500-fold. A point mutation in the γ - aminobutyric

acid (GABA) receptor can confer an increased resistance to cyclodiene insecticides. This

has been found in a range of different insect species (Bloomquist, 1994). None of the two

last mechanisms have until now been identified in spider mites.

1.2.2 Resistance in Norway

During several years of acricides tests to ascertain resistance status in agricultural pests,

Stenseth (1965) used a two-spotted spider mite strain (K-Strain) from a cucumber

greenhouse in Hardanger to investigate for susceptibility to acaricides. It was found that

the two-spotted spider mite (T. urticae) was resistant to dicofol and parathion. That was

the first reported case of multi-resistant mite in Norway. In another investigation,

Fjelldalen & Stenseth (1962) reported that a two-spotted spider mite strain was collected

on roses, and the chemical parathion was used to control them. But before the selection,

the strain already had a fairly high frequency (20%) of resistant spider mites. This

number increased during the trials and at the end reached an LC50 estimate of 400 times

higher than in a susceptible strain. From that time onward, not much has been done on

this species in Norway.

12

2. MATERIALS AND METHODS

2.1 Strains

The experimental work was undertaken in the laboratory of the Entomology Department

at Bioforsk, NIAER (Norwegian Institute for Agricultural and Environmental Research),

Ås, from the autumn semester of 2006 to the following autumn semester of 2007. Three

laboratory strains of the two-spotted spider mite, T. urticae species (Table 2) were used.

They were cultured on dwarf french bean plants, Phaseolus vulgaris L. The strains were

maintained at 24º C, a photoperiod of 8 - 16 h (L: D), and 60 ± 5 % RH in culture room

and 40-60 ± 5 % RH in growth room respectively. Two of the strains used were

Norwegian (i.e. NOR strains) as tested populations against a German susceptible strain

(GSS) for the investigation.

Table 2.Strains of Tetranychus urticae Tested, origin and host plant

Strain Origin Host Plant

NOR 4 Norway Bean

NOR 5 Norway Bean

GSS Germany Bean

2.2 Miticides

Six miticides were used during the experiment with different mode of actions. They were

grouped as follow:

I. Two METI acaricides:

a. Fenazaquin (Pride Ultra SC), produced by Dow AgroSciences, with recommended

dosage of 200g / L dH2O.

b. Fenpyroximate (Ortus – Akari 5SC), produced by Nihon Noyaku, with recommended

rate of 53g / L dH2O.

13

II. Two mite growth inhibitors:

a. Hexythiazox (Nissuron – Hexygon, DF) produced by Nippon Soda Co. LTD., Japan,

with, recommended rate of 65g /100 L dH2O.

b. Clofentezine (Apollo - Ovation SC), produced by Irvita Plant Protection N. V., with

recommended rate of 500g / L dH2O.

III. Abamectin (Vertimec - Avid 0.15EC), produced by Syngenta Crop Production AG,

Switzerland, with recommended rate of 35g / 100 L dH2O.

IV. Spirodiclofen (Admiral 10EC), produced by bayer CropScience, with recommended

rate of 40g / 100L dH2O.

2.3 Bioassay Procedure

Untreated bean plant (P. vulgaris) leaves were excised from the plant and placed with

the upper side of the leaf on technical solid water agar (1.3 % agar) base in 90 mm

diameter Petri-dish. The leaves-surfaces in the dishes were demarcated with a sticky

insect-glue applied with the aid of a plastic syringe. This was done to prevent mites from

possible escape during the experimental period, and to allow their observation in a

defined area.

With a binocular microscope and a fine sable / pint brush, eight to ten adult females of

the two-spotted spider mite strains, T. urticae, were transferred unto the demarcated areas

of the leaves in each petri-dish. They were covered with a ventilated dish cover and

stored in a growth room for oviposition for 24 hours. If 20 or more eggs are laid, the

female adults are removed. The numbers of eggs were counted, and the petri-dishes

stored in the growth room for hatching. The larvae hatched from the eggs after 4-5 days

were counted and results recorded. They were then immediately treated by spray-

application with acaricide(s). The application of the pesticides was done with four

replicates per concentration of each chemical, and with four concentrations plus distilled

water sprayed as control. The concentration used for each chemical and the process of

data collection and average mortality are found. An example of a chart with the necessary

information can be seen in appendix 1.

14

The six chemicals that were used were diluted into solutions of formulated acaricides

each. Immediately after the completion of the larvae count, petri-dishes containing T.

urticae nymphs of equal ages (i.e. NOR4, NOR5, and GSS) were sprayed with aqueous

solution of miticide using a constant spray rate in a Burkard – computer controlled

sprayer (Burkard Manufacturing LTD., UK.) with equal nozzle sizes firmly attached in a

fume cupboard. See section 2.2 above for the different acaricides and their recommended

dosage used in this investigation. Each of the mite populations were treated with four

different dosages of miticides (i.e. 0.25, 0.50, 1.00 and 2.00) of the above mentioned

aqueous solutions. Distilled water was used as control. The above concentrations

produced 0-100 % mortality. After 24 hours, mortality was scored and assessed. In this

investigation, the criteria used to describe mites / nymphs as dead or alive were based on

the following: ( a) if the selected mites have movement problem or cannot move, they

were considered as dead (Welthy et al., 1988); (b) if nymphs possibly walked-off from

demarcated leaf area prior to spraying, they are regarded as dead (Knight et al., 1990) and

(c) if nymphs are still in the dormant stage, they are scored as dead, but if both nymphs

and adults have no problem with locomotion, they are scored as alive. The samples were

again placed in growth room for another 48 hours (i.e. 72 hours total) after which a

second mortality count was conducted. Only the mortality after 48 hours was utilized for

the calculation of LC50 for all of the pesticides.

2.4 Statistical calculations

The percent mortality was calculated by dividing the sum of the number of dead larvae

per concentration in 48hrs by the number of units counted three days (72 hrs.) after the

application of the chemical (i.e. % mortality = ∑ of tot. dead in 48hr / # of units). All

results given in % mortality were corrected by using the “Abbot’s correction analysis”

(Abbott, 1925) (i.e. Abbot corrected = a –b /a * 100). An example is seen in Appendix 2-

a, b and c in the appendix section. The LC50 values and slopes were estimated by the use

of probit analysis, and resistance factors (RF) calculated according to the below formula

RF =LC50 or LC90 of laboratory collected population / LC50 or LC90 of susceptible

population (GSS).

15

3. RESULTS

The virulent effect of the six acaricides used against the two strains of the two-spotted

spider mite, T. urticae, was examined by the spray application method (bioassay). The

average mortality rate observed in 48 hrs for the control never exceeded 6.82%.

Generally, resistance of the two populations (NOR4 and NOR5) was significantly

different in all of the acaricide treatments compared to the GSS. The individual analysis

showed that resistance differed between treatments negligibly, and that some of the

chemicals had serious effect on the strains more than the resistant population therefore

they showed no resistance (i.e. RF < 1) as detailed in Tables 3 – 8.

Table 3. Selection for resistance and susceptibility with fenazaquin, in population of

Tetranychus urticae strains

Strain na Slope ± SE LC50 (g / L)

(0.95 CL)

LC90 (g / L)

(0.95 CL)

RF50b RF90

b

GSS 528 0.122 ± 0.009 12.048

(11.034-13.162)

11.129

(10.058-12.309)

__ __

NOR4 654 0.119 ± 0.008 22.784

(20.949-25.101)

21.629

(19.684-24.165)

1.10 1.1

NOR5 328 0.076 ± 0.009 7.447

(5.318-9.412)

24.402

(20.948-29.677)

0.67 1.13

CL confidence limit a Number of nymphs; b

resistance factor (RF) = LC50 or LC90 of laboratory collected population / LC50 or

LC90 of susceptible population (GSS)

In table 3 above, NOR4 exhibited low level of resistance to fenazaquin with RF value of

1.10 (i.e. RF >1) compared to GSS. NOR5 showed no resistance to the chemical

compared with the control (GSS).

16

Fenazaquin

0,0

20,0

40,0

60,0

80,0

100,0

120,0

1 2 3 4 5

Cons

% m

orta

lity

NOR4 NOR5 GSS

Figure 2: Resistance monitoring in three Tetranychus urticae strains with doses of fenazaquin at different concentration rates as mentioned in table 3. In the figure above, blue = NOR4, maroon = NOR5 and cream = GSS. In fig. 2 above, NOR5 showed the highest mortality rate compared to GSS, while NOR4

exhibited low mortality compared to the resistance strain.

17

Table 4. Selection for resistance and susceptibility with fenpyroximate, in population of

Tetranychu urticae strains

CL confidence limit

a Number of nymphs; bresistance factor (RF) = LC50 or LC90 of laboratory collected population / LC50 or

LC90 of susceptible population (GSS)

When fenpyroximate was applied at the recommended rate against NOR4, the strain

showed no resistance to the acaricide. Evidently, the RF value of 0.62 at LC50 falls

below the reference strain RF value of 1. NOR5 also showed no resistance against the

chemical compared with GSS.

Strain na Slope ± SE LC50 (g / L)

(0.95 CL)

LC90 (g / L)

(0.95 CL )

RF50b RF90

b

GSS 626 0.043 ± 0.003 30.346

(27.556-33.499)

59.946

(54.519-66.99)

__ __

NOR4 474 0.08 ± 0.007 18.709

(16.877-20.795)

34.637

(31.211-39.364)

0.62 0.58

NOR5 370 0.045 ± 0.005 22.743 (19.507-

26.301)

51.046

(44.966-59.714)

0.75 0.85

18

Fenpyroximate

0,0

20,0

40,0

60,0

80,0

100,0

120,0

1 2 3 4 5

Cons

% m

orta

lity

NOR4 NOR5 GSS

Figure 3: Resistance monitoring in three Tetranychus urticae strains with doses of fenpyroximate at different concentration rates as mentioned in table 4. In the figure above, blue = NOR4, maroon = NOR5 and cream = GSS. In fig. 3, NOR4 strain showed the highest mortality rate at the recommended dosage

compared to the resistance strain GSS because the lower the RF value as shown in tab. 4,

the higher the mortality. NOR5 showed a higher mortality rate at the same recommended

dose of fenpyroximate application compared with GSS. See table 4 above.

19

Table 5. Selection for resistance and susceptibility with hexythiazox, in population of

Tetramychus urticae strains

CL Confidence limit aNumber of nymphs; b Resistance factor (RF) = LC50 or LC 90 of laboratory collected population / LC50 or

LC90 of susceptible population (GSS)

In the trial, NOR4 showed a little more of resistance factor (RF) value of 1.20 at LC50

when hexythiazox was used to control it compared with the GSS value of 1. The NOR5

strain also exhibited a slightly higher level of resistance to hexythiazox with RF value of

1.24 compared with GSS.

Strain

ηa

Slope ± SE

LC50 ( g/100L)

(0.95CL)

LC90 ( g/100L)(0.95CL)

RF50 b

RF90 b

GSS 633 17.592 ± 1.382 0.087 (0.079-0.095)

0.159 (0.146-0.178)

__ __

NOR4 429 9.884 ± 0.895 0.103 (0.088-0.118)

0.232 (0.207-0.267)

1.20 1.47

NOR5 453 10.678 ±0.950 0.108

(0.095-0.122) 0.228

(0.205-0.259) 1.24 1.43

20

Hexythiasox

0,0

20,0

40,0

60,0

80,0

100,0

120,0

1 2 3 4 5

Cons

% m

orta

lity

NOR4 NOR5 GSS

Figure 4: Resistance monitoring in three Tetranychus urticae strains with doses of hexythiazox at different

concentration rates and a control as mentioned in table 5. In the figure above, blue = NOR4, maroon =

NOR5 and cream = GSS.

In figure 4, both NOR4 and NOR5 showed a lower mortality rate then the GSS strain

when treated with hexythiazox.

21

Table 6.Selection for resistance and susceptibility with clofentezine, in population of Tetranychus

urticae strains

Strain na Slope ± SE LC50 (g / L) (0.95 CL)

LC90 (g / L) (0.95 CL)

RF50b RF90

b

GSS 518 0.084 ± 0.008 15.44 (13.833-17.413)

30.539 (27.144-5.299)

__ __

NOR4 562 0.066 ± 0.007 22.33 (19.736 25.998)

41.687 (36.046-0.295)

1.45 1.37

NOR5 480 0.071 ± 0.008 19.061

(16.924-21.88) 37.196 (32.459- 4.282)

1.23 1.22

CL confidence limit, a Number of nymphs; bresistance factor (RF) = LC50 or LC90 of laboratory collected population / LC50 or

LC90 of susceptible population (GSS)

In table 6 above, it was observed that at LC50, the NOR4 strain showed a little higher

level of resistance to clofentezine with RF value of 1.45 compared with GSS. NOR5 also

exhibited a little more of resistance with RF value of 1.23 compared to GSS.

22

Clofentezine

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

1 2 3 4 5

Cons

% m

orta

lity

NOR4 NOR5 GSS

Figure 5: Resistance monitoring in three Tetranychus urticae strains with doses of clofentezin at different concentration rates as mentioned in table 6. In the figure above, blue = NOR4, maroon = NOR5 and cream = GSS.

Figure 5 showed that the mortality rate for NOR4 was lower than GSS. Similarly the

mortality rate for NOR5 was lower than the control (GSS). The result shown in tab. 6

implies that both NOR4 and NOR5 were resistant to clofentezine than the control

because their RF values were higher than GSS.

23

Table 7. Selection for resistance and susceptibility with abamectin, in population of Tetranychus

urticae strain

CL confidence limit a Number of nymphs; b resistance factor (RF) = LC50 or LC90 of laboratory collected population / LC50 or

LC90 of susceptible population (GSS)

The Norwegian strain 4 (NOR4) showed a resistance factor (RF) value that is twice more

than the control strain (GSS) when abamectin was used to control it. Conversely, NOR5

showed a high level of resistance to abamectin with RF value of 1.69 compared with the

GSS strain.

Strains

na

Slope ± SE

LC50 (g /100L) (0.95 CL)

LC90 (g / 100L) (0.95 CL)

RF50b

RF90

b

GSS 589 0.034 ±0.003 26.451

(22.272-30.452)

63.983

(57.954-71.817)

__ ___

NOR4 498 0.018 ±0.002 54.371

(47.191-61.962)

126.974

(113.474-145.419)

2.06 1.10

NOR5 363 0.033 ±0.003 44.699

(39.352-50.466)

83.722

(75.207-95.277)

1.69

1.31

24

Abamectin

0,0

20,0

40,0

60,0

80,0

100,0

120,0

1 2 3 4 5

Cons

% m

orta

lity

NOR4 NOR5 GSS

Figure 6: Resistance monitoring in three Tetranychus urticae strains with doses of abamectin at different

concentration rates as mentioned in table 7. In the figure above, blue = NOR4, maroon = NOR5 and cream

= GSS.

The result in fig. 6 above corresponds with the resistant of NOR4 to abamectin shown in

tab. 7. The mortality rate of the strain was lowest compared with GSS when the

recommended dosage of the pesticide was applied. This implies that it was more resistant

to the chemical. NOR5 showed a lower mortality rate than the control against the same

pesticide due to its high resistant rate.

25

Table 8. Selection for resistance and susceptibility with spirodiclofen, in population of

Tetranychus urticae strains

CL Confidence limit a Number of nymphs; bresistance factor (RF) = LC50 or LC90 0f laboratory collected population / LC50 or

LC90 of susceptible population (GSS)

In table 8, NOR4 showed no resistance to spirodiclofen with RF value of 0.83 compared

to the GSS. NOR5 equally showed no resistance with RF value of 0.83 at the same

concentration compared to the control.

Strain

na Slope ± SE LC50 (g / 100L)(0.95 CL)

LC90 (g / 100L(0.95 CL)

RF50b RF90

b

GSS

522 41.736 ± 10.196 0.012 (0.009-0.017)

0.042 (0.031-0.075)

__ __

NOR4 495 58.903 ± 10.395 0.01 (0.008-0.013)

0.032 (0.025-0.045)

0.83 0.76

NOR5 373 48.345 ± 11.776 0.01

(0.008-0.015) 0.037

(0.027-0.064) 0.83 0.88

26

Spirodiclofen

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

90,0

100,0

1 2 3 4 5

Cons

% m

orta

lity

NOR4 NOR5 GSS

Figure 7: Resistance monitoring in three Tetranychus urticae strains with doses of spirodiclofen at different concentration rates as mentioned in table 8. In the figure above, blue = NOR4, maroon = NOR5 and cream = GSS. In figure 7, both NOR4 and NOR5 showed higher level of mortality to spirodiclofen

when it was used against them compared with GSS. This fact is shown in the result in

tab. 8 above where both strains exhibited no resistance (RF < 1) against the chemical at

the same concentration rate.

27

4. DISCUSSIONS A standard bioassay is conducted, according to Nauen et al., 2001, by spray application

of the spider mite larvae. The method is used because it is one of the best ways of

investigating the effect of foreign substances such as drugs in living organisms when

little is known about the mechanism. The potency of acaricides differs with mite species.

The effectiveness tests of the six pesticides on two Norwegian strains of the two-spotted

spider mite T. urticae Koch (Acari: Tetranychidae) show difference in resistance. They

revealed a vast range of no and low levels of resistance in the acaricides. Both strains

exhibit low level of resistance to the mite growth inhibitors. For the METI acaricides,

they showed susceptibility or little resistance.

The two acaricides, fenazaquin and fenpyroximate, are well known METI (mitochondrial

electron inhibitor) acaricides, and are potent foreign substances that function primarily by

hindering complex I (NADH: ubiquinone oxidoreductse) of the mitochondrial respiratory

chain (Hollingworth & Ahammadsahib, 1995). They are said to be widely used because

of their high potency against many mite species. However, some strains of Tetranychus

spp from Australia – T. urticae Koch (Herron et al., 1993) and Japan – T. kanzawai

Kishide (Goka, 1998) have been reported to show over 100-fold resistance to the

compounds. Furthermore, two UK strains (TUK4 and TUK5) used in a study by Devine

et al. (2001) were said to be the first for which control failures for METI – acaricides in

Europe have been confirmed. It is therefore clear from the above studies and others that

the effectiveness of METI – acaricides is at risk, particularly where the use of pesticides

interacts with ecological and genetic factors which encourage the development of

resistance (Devine et al., 2001). In this investigation, both Norwegian strains of the two-

spotted spider mite (NOR4 and NOR5) showed no resistance to fenpyroximate. NOR4

exhibited low level of resistant to fenazaquin, while NOR5 showed no resistance in the

bioassay. The resistance factor value at LC50 for NOR4 when fenpyroximate was applied

against it was 0.62, while that of NOR5 was 0.75 indicating that the GSS had a higher

tolerance. With regards to fenazaquin, NOR4 showed a little higher level of resistance

(RF value of 1.10) at LC50 which is greater than GSS, whilst NOR5 showed no resistance

28

(RF 0.67 < 1) at LC50. The moderate level of resistance of NOR4 to fenazaquin may have

evolved from its ability to speedily adapt to pesticides when used against it for some

time. The susceptibility of NOR5 cannot easily be explained. Since the strains were

constantly kept under greenhouse condition, their adaptation to the condition as opposed

to field condition could also contribute to the resistance of NOR4 to fenazaquin, and the

no resistance of both NOR4 and NOR5 to fenpyroximate observed.

.

Hexyhtiazox and clofentezin cause little or no loss of life to adult female spider mite, but

they act as limitation to their laying of many fertile eggs (Chapman & Marris, 1986).

Nymphs hatched from the affected eggs do not undergo normal molting due to the

disruption of chitin synthesis (Flexner et al., 1995; Dekeyser & Downer, 1994). However,

resistance to these chemicals has been noticed in both the European red mite and the two-

spotted spider mite (Nauen et al., 2001). In the current investigation, both NOR4 and

NOR5 show low level of resistance to hexythiazox and clofentezin as seen in Tables 5

and 6. The resistance is due to the strains been capable of adjusting to the effect of the

mite growth inhibitor acaricides. Yet, nothing can be said concerning the mechanisms

resulting in T. urticae resistance against these acaricides (Stumpf, 2001).

Abamectin acts as nematicide, acaricide and insecticide (Putter et al., 1981). It acts on the

γ - aminobutyric acid (GABA) and glutamate-gated chloride channels, leading to

vigorousness of the chloride ion channel at higher concentrations and paralysis of the

pests (Bloomquist, 2001; Fritz et al., 1979). Spider mite, especially the two-spotted spider

mite, T. urticae, are the main aim of abamectin, and it is one of the most potent chemicals

hostile to the nymphs of a susceptible T. urticae strain (Nauen et al., 2001). This

investigation shows NOR4 and NOR5 strains to exhibit a resistance factors that were

twice more and very higher than the susceptible GSS population as seen in Table 7. The

highest resistant in the above strains may be due to the ability of spider mites to speedily

adjust to xenobiotic when applied against them. Stumpf & Nauen (2001), state that these

biological mechanisms are difficult to examine and almost nothing is known regarding

the mechanisms conferring abamectin resistance. Nauen et al. (2001) elucidate that T.

urticae are major target organisms of abamectin; but Compos et al. (1995), and Compos

29

et al. (1996) say resistance to abamectin in T. urticae is known from California, Florida

and the Netherlands. Furthermore, Stumpf et al. (2001) explain that the genetically

established resistance mechanisms in spider mites were similar to those found in insects,

and has increased degeneration of acaricides through esterases, glutathione S-

transferases (GSTs), or cytochrome P450 – dependent monooxygenases (MFO) (metabolic

resistance), and molecular changes in the target site, so that the target is no longer

sensitive to pesticide inhibition (target site resistance). Note that the two strains originate

from the greenhouse where they may have adapted to diverse conditions over a period of

time.

Spirodiclofen is one of the many new pesticides that are effective against all growth

stages and mature female of the tetranychid mite such as the two-spotted spider mite,

Tetranychus urticae. It is said not to exhibit cross-resistance to OP’s, METI’s,

hexythiazox and abamectin in well-distinguished species with high resistance to at least

one of the above chemicals (Rauch & Nauen, 2003). As a member of the tetronic acid

acaricide group, it has no adverse effect on insects of benefit and causes little harm to

predatory mites; it is different from some other pesticides in that it has short milieu

staying power (DT50 0.5-5.5 days) and a low mammalian injury (Fischer et al., 1993;

Wachendorff et al., 2000; Nauen et al., 2002). The two Norwegian strains (NOR4 and

NOR5) show no resistance when spirodiclofen was used against them.

5. Conclusion

The resistance in the two Norwegian populations of the two-spotted spider mite

(Tetranychus urticae) (NOR4 and NOR5) for the six selected acaricides (see section 2.2)

vary contingent on their mode of actions compared with the resistance strain (GSS).The

average mortality obtained after 48 hours in the control was 6.82 %.

Strain NOR4 exhibited low level of resistance to fenazaquin with RF value of 1.10

compared with GSS, and NOR5 showed no resistance to the poison with RF value of

0.67. Both NOR4 and NOR5 exhibited no resistance to fenpyroximate with RF values of

30

0.62 and 0.75 respectively compared with GSS. The resistance factor (RFs) for NOR4

and NOR5 strains when hexythiazox was applied were at low level (i.e. 1.18 and 1.24)

with respect to GSS. NOR4 and NOR5 showed low level of resistance factor of 1.45, and

1.23 respectively to clofentazine than GSS. When abamectin was used in the trial, the Rf

value for NOR4 strain was two times more than the reference strain GSS, with a resistant

factor of 2.06 which was the highest resistance of the six meticides. NOR5 strain gave a

moderate level of RF value of 1.69 at LC50 compared with GSS. NOR4 and NOR5

showed no resistance against spirodiclofen when it was applied with RF values of 0.83

and 0.83 respectively. Both NOR4 and NOR5 showed high resistant to abamectin whilst

they exehibited no resistanc to spirodiclofen and fenpyroximate.

One can therefore suggest that the acaricides used in this trial, if used to control the two-

spotted spider mite, should be used in combination with other insecticides, or should not

be used at tall. For instance, fenpyroximate and spirodiclofen could be used with their

exact recommended rates or be included in integrated pest management (IPM)

programmes for increased potency. Biological control method could be an alternative

way of controlling the two spotted spider mite. For instance; the use of Thrips of various

species which are considered as most important predators of spider mites and eggs (Van

de Vrie et al., 1972), and the use of natural enemy fungi like Neozygites floridana that

kills the two spotted spider mite (Klingen & Westrum, 2007).

31

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7. APPENDIX Appendix 1.Example of a chart showing concentrations, the process of data collection and averages

Strain B Fenazaquin

NOR 5 EGG LARVEADEAD 24 HOUR

DEAD 48 HOUR

% DEAD 24 HOURS

% DEAD 48 HOURS AVERAGE

cons. A 25 12 1 2 8 16.7 21.4 0 B 20 20 1 3 5 15.0 C 26 13 0 3 0 23.1 D 22 13 1 4 8 30.8 0.25 A 27 19 3 4 16 21.1 31.5 B 20 14 3 6 21 42.9 C 30 14 2 5 14 35.7 D 27 19 4 5 21 26.3 0.5 A 37 15 3 7 20 46.7 57.5 B 20 12 6 8 50 66.7 C 31 13 5 9 38 69.2 D 25 19 7 9 37 47.4 1 A 27 15 8 15 53 100.0 83.1 B 22 22 10 14 45 63.6 C 35 10 10 10 100 100.0 D 21 16 9 11 56 68.8 2 A 37 20 12 12 60 60.0 85.0 B 20 16 13 14 81 87.5 C 30 19 15 19 79 100.0 D 36 27 14 25 52 92.6

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Appendix 2: Examples of Abbot’s correction (a-c) Appendix 2-a

Strain C Spirodiclofen Abbot GSS EGG LARVEA DEAD 24

HOUR DEAD 48 HOUR

% DEAD 24 % DEAD 48 AVERAGE corrected

cons. A 29 25 2 3 8 12.0 13.9 0 B 30 24 2 4 8 16.7 C 40 30 1 4 3 13.3 D 36 22 0 3 0 13.6 0.25 A 28 21 2 5 10 23.8 25.2 13.1 B 36 34 4 4 12 11.8 C 33 28 2 8 7 28.6 D 40 19 3 7 16 36.8 0.5 A 35 23 5 11 22 47.8 42.0 32.6 B 37 30 7 14 23 46.7 C 31 24 5 8 21 33.3 D 39 25 6 10 24 40.0 1 A 28 19 4 12 21 63.2 50.5 42.5 B 35 29 8 14 28 48.3 C 40 32 9 13 28 40.6 D 33 32 10 16 31 50.0 2 A 30 28 10 23 36 82.1 84.7 82.2 B 28 17 12 17 71 100.0 C 40 28 11 22 39 78.6 D 37 32 17 25 53 78.1

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Appendix 2-b Abbot Tot. Tot. Tot. Not Mortality % not AVERAGE corrected treated dead dead % dead 13.9 101 14 87 13.9 86.1 25.2 13.1 102 24 78 25.2 74.8 42.0 32.6 102 43 59 42 58 50.5 42.5 112 55 57 50.5 49.5 84.7 82.2 105 87 18 84.7 15.3

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Appendix 2-c Mort Dose Mort. Average 0 13.9 0.25 13.1 0.25 25.2 0.5 32.6 0.5 42 1 42.5 1 50.5 2 82.2 2 84.7