Pieter_Van_Nieuwenhuyse-PhD_dissertation-20121210

MMiittoocchhoonnddrriiaallllyy eennccooddeedd aaccaarriicciiddee rreessiissttaannccee
iinn ssppiiddeerr mmiitteess
Pieter Van Nieuwenhuyse
ISBN 978-90-5989-569-0
Mitochondriallyencodedacaricideresistance
inspidermitesPieterVanNieuwenhuyse2012

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“Le coeur a ses raisons que la raison ne connaît pas”
(Blaise Pascal, 1623-1662)

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Promoters: Prof. dr. ir. Luc Tirry
Laboratory of Agrozoology
Department of Crop protection
Faculty of Bioscience Engineering
Ghent University
Prof. dr. ir. Bartel Vanholme
Department of Plant Systems Biology
Flanders Institute for Biotechnology (VIB-UGent)
Department of Plant Biotechnology and Bio-informatics
Faculty of Science
Ghent University
Dr. ir. Thomas Van Leeuwen
Laboratory of Agrozoology
Department of Crop protection
Faculty of Bioscience Engineering
Ghent University
Dean: Prof. dr. ir. Guido Van Huylenbroeck
Rector: Prof. dr. Paul Van Cauwenberge

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Mitochondrially encoded acaricide resistance
in spider mites
ir. Pieter Van Nieuwenhuyse
Thesis submitted in fulfillment of the requirements for the degree of
Doctor (PhD) in Applied Biological Sciences

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Dutch translation of the title:
Mitochondriaal gecodeerde acaricideresistentie in spintmijten
Front cover: (Up) Compilation of a wordle cloud, generated from the text in this dissertation using
the toy WordleTM
(www.wordle.net), a schematic picture of a cell containing the mitochondria and
their mtDNA molecules (www.genome.gov), and structural formulae of bifenazate (up left) and
acequinocyl (down right) respectively. (Below) Banner of pictures of adult females from four spider
mites species (from left to right): Tetranychus urticae (picture by Jarmo Holopainen), T. pacificus
(www.iv.ucdavis.edu), Panonychus ulmi (www.escalera.bio.ucm.es) and P. citri
(www.agrolink.com.br)
Please refer to this work as follows:
Van Nieuwenhuyse, P (2012) Mitochondrially encoded acaricide resistance in spider mites. PhD
thesis. Ghent University, Ghent, Belgium.
ISBN 978-90-5989-569-0
This study was funded by grant number SB073454 from the Institute for the Promotion of
Innovation through Science and Technology in Flanders (IWT Vlaanderen).
The author and the promoters give the permission to use this thesis for consultation and to copy
parts of it for personal use. Every other use is subject to the copyright laws, more specifically the
source must be extensively specified when using results from this thesis.

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Members of the examination committee
Prof. dr. ir. Luc Tirry
Promoter
Department of Crop Protection,
Faculty of Bioscience Engineering,
Ghent University
Prof. dr. ir. Bartel Vanholme
Promoter
Department of Plant Biotechnology and Bioinformatics,
Faculty of Science,
Ghent University
Department of Plant Systems Biology,
VIB
Dr. ir. Thomas Van Leeuwen
Promoter
Ghent University
Prof. dr. ir. Marc Van Meirvenne
Chairman
Department of Soil Management,
Ghent University
Prof. dr. Godelieve Gheysen
Secretary
Department of Molecular Biotechnology,
Ghent University
Prof. dr. ir. Patrick De Clercq
Ghent University
Prof. dr. John Vontas
Department of Biology,
University of Crete,
Greece
Dr. Shuvash Bhattarai
Evesham Technology Centre,
Chemtura Europe Ltd.,
UK
Dr. Ralf Nauen
Research Insecticides,
Insect Toxicology and Resistance,
Bayer CropScience AG,
Germany

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Woord vooraf – Dankwoorden…………………………….……………………………………..
Het boekje dat u momenteel aan het doorbladeren bent, was er niet gekomen zonder de hulp van
vele mensen. Eerst en vooral wil ik mijn promoters bedanken. Luc, bedankt dat je mij de kans gaf
om in het “beestjeslabo” mijn onderzoek te starten. Bartel, jou ben ik heel veel verschuldigd voor
jouw introductie in de moleculaire wereld, jouw gemoedelijkheid en bereidheid om op elk mogelijk
tijdstip een luisterend oor en als wandelend encyclopedie een raadgever te zijn. Heel erg jammer dat
je zo vroeg de faculteit verliet. Thomas, als “master head” van het labo Agrozoölogie bedank ik je
heel erg voor de intense begeleiding en de sturende werking, in goeie en minder goeie momenten.
De uitvalsbasis van het dagelijks werk bevond zich in een bureautje helemaal op het einde
van de gang, zowaar een beetje verstopt achter de branddeur. Op die plaats heb ik heel fijne mensen
ontmoet, in het begin en ook later. Katrien, bedankt om “partner in crime” te zijn. Bedankt om jou
levenservaringen en –overtuigingen met mij te delen, en om vele rondjes met mij rond de
Watersportbaan te lopen. Het was een eer om jouw bureaumaatje te zijn. Uiteindelijk verkaste je
naar een andere werkvloer, maar de indruk die je bij mij naliet is nooit verdwenen. Fernando, I
thank you for bringing the Spanish sun in our office and for joining me during several running
sessions. Jisheng, thanks for your smile every day and your stories about the Chinese traditions and
teas. Good luck with your research! Bieke, jouw verblijf bij ons was helaas van korte duur, maar de
babbels met jou waren altijd oprecht en verrijkend, bedankt daarvoor. Dorien, ik heb je naam zoveel
keren verkeerd uitgesproken: alleen al voor jouw begrip daarvoor zou ik je heel dankbaar moeten
zijn. Maar ik bedank je vooral voor je opgewektheid, jouw muzikaliteit en directheid. Ivan, bedankt
voor de vele gedachtewisselingen over wetenschappelijke onderwerpen, en ook voor de gesprekken
van totaal andere aard. Peter, jij was niet alleen lid van ons bureau, je was vooral een heel
betrouwbare en behulpzame collega in het mijtenwerk. We begonnen samen in het “radioactief
laboratorium” en je leverde meteen schitterend werk, iets wat een constante bleek voor de rest van
de daaropvolgende jaren. Ik ben je ongelooflijk dankbaar.
In de rij van de “mijten”-collega’s bedank ik Steven voor de intense samenwerking binnen
de “boerekot”muren - zowel in de mijtenkelder als in het labo - en voor de loopsessies/activiteiten
ver buiten deze muren, Wannes voor de raad en daad bij verwerking van gegevens en voor de trip
naar Brazilië, Nicky voor de opgewekte opgestoken hand bij de gang-ontmoetingen en jouw hulp
bij de grafische verwerking, Astrid en Annelies voor jullie leuke babbels. Jahangir, I profoundly
thank you for your intense collaboration and kindness during your stay in our lab. I could not have
done all the mite work without your help.
Ik ben ook alle (oud-)collega’s van het laboratorium Agrozoölogie heel dankbaar, en niet in
het minst Veronic, Nick en Maarten om diverse redenen. Bovendien wil ik Olivier en Yves extra

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bedanken voor onze gezamenlijke fietstochtjes. Thijs, jou bedank ik speciaal voor je empathie, je
bereidheid om paraat te staan voor collega’s en je raad en daad.
Ook de ondersteuning door ons “technisch comité” ben ik zeer erkentelijk. Leen, merci voor
de babbels. Rik, een oprecht dankjewel voor je talloze interventies en gesprekken over heel brede
thema’s, waaronder Australië. Pipier, bedankt om mij voortdurend Dieter te noemen.
Ik heb ook mooie mensen in andere labo’s ontmoet. Joachim, bedankt voor je wilde
uitspattingen, je interessante levenswijsheden en je hulp bij het betere “gel”-werk. Dankzij jou werd
het minder “tsjolen”. Isabel, een oprecht merci voor je hulp en uitleg op zovele vlakken. Maarten,
dank voor jouw medewerking omtrent het massaspectrometriewerk, hopelijk was het voor jou ook
nuttig ;o). Samuel en Erik, bedankt dat we jullie labo mochten gebruiken om radioactieve mijten
verder te verwerken. Aina, je te remercie pour tes efforts dans l’exploration de la recherche
écologique.
Geen thesis zonder vrienden. Ik zou hier een lijstje kunnen opsommen van hele mooie
mensen, maar ik ben bang dat ik er toch nog zal vergeten en dat kan niet de bedoeling zijn. In de
plaats zeg ik aan hen de woorden van Cicero: “Zonder vriendschap is het leven niets waard”.
Daarnaast voelde ik mij de voorbijgaande jaren gesteund door mijn directe familie. Pa en
ma, bedankt om mij niet aflatend te vragen hoe het met mij ging en hoe ik het doctoraat ging
afwerken. Mijn zussen Liesje en Swito, bedankt voor jullie babbels en steun de afgelopen jaren.
Zonder jullie was het boekje er niet gekomen. Ook Cemiel en Sampie betrek ik in de hulde. Unc,
bedankt voor je sarcastische opmerkingen op tijd en stond. Ook moet ik– tegen heug en meug,
grapje! - mijn schoonfamilie in spe bedanken voor de niet-wetenschappelijke ondersteuning ;o).
Kennis is stuurloos zonder liefde. Olivia, mijn Sjabolleke, ongelooflijk bedankt voor alles.
Simpelweg alles.
Pieter, december 2012

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Abbrevations ..................................................................................................................................................
a.i. active ingredient
ACCase acetyl-coenzyme A carboxylase
AChE acetylcholinesterase
ANOVA
CARB
Analysis of Variance
carbamate
COE carboxylesterase
P450 cytochrome P450 dependent monooxygenase
cytb cytochrome b
cytc1
DDT
cytochrome c1
dichlorodiphenyltrichloroethane
DEF S,S,S-tributylphosphorotrithioate
DEM diethylmaleate
DNA deoxyribonucleic acid
EDTA
GABA
ethylenediaminetetraacetic acid
γ-amino butyric acid
GSS German susceptible strain
GST glutathione-S-transferase
IC50 concentration at which 50% inhibition is realised
IMS intermembrane space
IPM integrated pest management
IRAC Insecticide Resistance Action Committee (www.irac-online.org)
ISP
LC50
Rieske iron-sulfur protein
concentration at which 50% of animals is killed
LS-VL laboratory susceptible strain
METI mitochondrial electron transport inhibitor
MR-VL multiresistant strain
mtDNA mitochondrial DNA
nAChR nicotinic acetylcholine receptor
NCBI
OP
National Center for Biotechnology Information
organophosphate
PBO piperonylbutoxide
PCR
ppm
QoI
Polymerase Chain Reaction
parts per million, mg active ingredient per litre
ubiquinol oxidizing site inhibitor

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RR resistance ratio
SC suspension concentrate
SDS
SEM
sodium dodecyl sulfate
standard error of the mean
SR synergistic ratio

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CONTENT………………………………………………………………………………………………………………………………………………………………
Scope and thesis outline I.
CHAPTER I. INTRODUCTION TO SPIDER MITE CONTROL 1
1. Tetranychidae (spider mites) 2
1.1 Classification, biology and agricultural relevance 2
1.2 Integrated control of spider mites 8
1.2.1 Cultivation measures to control spider mites 9
1.2.2 Biological and physical control of spider mites 9
1.2.3 Chemical control of spider mites 11
2. Mitochondria as targets of acaricides 13
2.1 Mitochondrial electron transport (MET) 13
2.1.1 Oxidative phosphorylation (OXPHOS) chain 13
2.1.2 Mitochondrial genomes 16
2.1.2.1 Biology of mitochondrial genomes and mutagenesis 16
2.1.2.2 Biological and clinical relevance of mitochondrial genomes 19
2.1.2.3 Mitochondrial genomes of insects and (spider) mites 21
2.1.3 Complex III and the protonmotive Q cycle 24
2.2 Acaricides targeting nuclearly encoded proteins of mitochondrial OXPHOS complexes I, II, IV and V 27
2.3 Chemicals targeting mitochondrially encoded cytb protein of OXPHOS complex III 28
2.3.1 Non-insecto-acaricidal inhibitors of mitochondrially encoded cytb 28
2.3.2 QoI insecto-acaricides (Insecto-acaricidal inhibitors of mitochondrially encoded cytb Qo) 32
2.4 Bifenazate 33
3. Spider mite resistance to insecto-acaricides 34
3.1 Resistance: definition, origin, detection, genetics and magnitude 35
3.2 Nuclearly encoded resistance 38
3.2.1 Resistance mechanisms in insects/spider mites to insecto-acaricides targeting nuclearly encoded
protein s 38
3.2.1.1 Target site resistance 39
3.2.1.2 Metabolic resistance 39
3.2.1.3 Cross- and multi-resistance 40
3.2.2 Spider mite resistance to inhibitors of OXPHOS complexes I and V 40
3.3 Mitochondrially encoded resistance 41
3.3.1 Putative mitochondrially encoded resistance to pyrethroids and avermectins in insects 42
3.3.2 Resistance to QoIs in organisms excluding insects and mites 42

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CHAPTER II.BIFENAZATE RESISTANCE: LINK WITH MUTATED MITOCHONDRIALLY
ENCODED CYTB QO AND PARALLEL EVOLUTION IN SPIDER MITES 47
1. Maternally inherited bifenazate resistance in Tetranychus urticae and parallel bifenazate resistance evolution
in closely related Panonychus citri 48
1.1 Introduction 48
1.2 Materials and methods 49
1.2.1 Spider mite strains 49
1.2.2 Chemicals 49
1.2.3 Bioassays 50
1.2.4 Crossing experiments 50
1.2.5 DNA extraction 51
1.2.6 Amplification (PCR), sequencing and cytb genotyping 51
1.3 Results 53
1.4 Discussion 58
1.5 Conclusions 62
2. Cytb mediated bifenazate resistance is not correlated with compensatory mutations in the ISP and cytc1 in
Tetranychus urticae 62
2.1 Introduction 62
2.2.1 Strains 63
2.2.2 RNA extraction, DNA synthesis, PCR and sequencing 63
2.3 Results 64
2.4 Discussion and conclusions 65
CHAPTER III. GENETIC MITOCHONDRIAL BOTTLENECK, HETEROPLASMY AND
REPERCUSSIONS FOR BIFENAZATE RESISTANCE IN T. URTICAE 67
1. Introduction 68
2. Materials and Methods 68
2.1 Strains 68
2.2 PCR, cloning, sequencing and quantification of heteroplasmy 69
2.3 Establishing mutation threshold for phenotypic expression of resistance 71
2.4 Modeling the inheritance of heteroplasmy 71
3. Results 72
3.1 Quantification of mitochondrial heteroplasmy and inheritance 72
3.2 Establishing mutation threshold for phenotypic expression of resistance 76
3.3 Modeling the inheritance of heteroplasmy 76

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4. Discussion 77
5. Conclusions 81
CHAPTER IV. QOI CROSS-RESISTANCE AND CURRENT FIELD STATUS IN SPIDER MITES
83
1. Cytb mutations conferring cross-resistance between QoIs in the two-spotted spider mite Tetranychus urticae
and the citrus red mite Panonychus citri 84
1.1 Introduction 84
1.2.1 Mite strains 85
1.2.2 Cytb sequence 85
1.2.3 Chemicals 85
1.2.4 Toxicity bioassays 85
1.2.5 Crossing experiments 85
1.2.6 Enzyme assays and synergism tests 86
1.3 Results 87
1.3.1 Resistance to bifenazate, fluacrypyrim and acequinocyl 87
1.3.2 Cytb genotype in WI and BEL1 90
1.3.3 Mode of inheritance of acequinocyl and fluacrypyrim resistance 90
1.3.4 Synergism 94
1.3.5 Detoxifying enzyme activity 95
1.4 Discussion 96
1.5 Conclusions 102
2. Current status of QoI field resistance in Tetranychus urticae populations collected from Belgium and the
Netherlands 103
2.1 Introduction 103
2.2.1 Mite strains 103
2.2.2 Cytb and partial COI sequence 104
2.2.3 Chemicals 104
2.2.4 Toxicity bioassays 104
2.2.5 Enzyme assays 104
2.3 Results 105
2.3.1 Resistance to bifenazate and acequinocyl 105
2.3.2 Cytb genotype and partial COI sequence 107
2.3.3 Detoxifying enzyme activity 108
2.4 Discussion 109
2.5 Conclusions 111

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3. Bifenazate resistance in field strains of Tetranychus urticae collected from Australia and Tetranychus pacificus
collected from California not mediated by cytb amino acid subsitutions 112
3.1 Introduction 112
3.2.1 Strains 112
3.2.2 DNA extraction 113
3.2.3 Primers 113
3.2.4 PCR and sequencing 114
3.3 Results 114
3.4 Discussion and conclusions 115
CHAPTER V. BIFENAZATE MODE OF ACTION 119
1. Introduction 120
2. Materials and methods 121
2.1 Mites and insects 121
2.2 Chemicals 121
2.3
14
C-labeled bifenazate 121
2.4 Liquid chromatography–mass spectrometry (LC-MS) 122
2.5 Radiolabeling of mites and insects 122
2.6 Isolation of mitochondria and preparation of mitochondrial extracts 123
2.7 Two-dimensional Blue Native/Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (PAGE) 123
2.8 Bifenazate metabolism assays and Thin Layer Chromatography 124
2.9 Genotyping of GABA-gated chloride channel (GABACl) genes in bifenazate resistant and susceptible strains
125
2.10 Quantitative real-time PCR (qPCR) 126
3. Results 127
3.1 Two-dimensional Blue Native/Sodium Dodecyl Sulfate PAGE of mitochondrial extracts from treated mites 127
3.2 Bifenazate metabolism and Thin Layer Chromotography 131
3.3 GABACl genotyping and relative gene expression in bifenazate resistant and susceptible strains 133
4. Discussion 134
5. Conclusions 137
CHAPTER VI. CONCLUSIONS AND PROSPECTS 139
Summary 151

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Samenvatting 157
Addendum 163
References 167
Curriculum vitae 195

························································································Scope and thesis outline
II.
The phytophagous mite family of spider mites (family Tetranychidae) harbours mite species that are
associated with major losses in several agricultural crops worldwide. Spider mites are commonly
controlled by chemical treatments with miticides as part of integrated pest management, however,
resistance to all classes of miticides develops rapidly after introduction in field which compromises
severly an efficient control of these pest organisms. Resistance development has also been reported
for the recently worldwide commercialised carbazate acaricide bifenazate with current unknown
mode of action. In this study we have tried to unravel the mechanisms that underlie the resistance
against bifenazate at the toxicological, genetic, molecular and biochemical level, which could
indirectly elucidate more aspects about bifenazate’s mode of action.
Chapter 1 highlights the remarkable biological properties of spider mites rendering them quickly
insensitive to acaricides of any mode of action class using different resistance mechanisms.
Mitochondria are discussed as targets of acaricides with a special attention for complex III and its
Q-cycle, the unique features of mitochondrial genomes and consequences for resistance
development.
In the light of resistance to bifenazate in spider mites, we discussed previously reported parallel
genetic adaptations correlated with mitochondrially encoded target site resistance to chemicals in
pathogenic fungi and protozoans.
In Chapter 2 it is shown high that bifenazate resistance independently and parallelly evolved in
several laboratory and field strains of the twospotted spider mite Tetranychus urticae and the citrus
red mite Panonychus citri and that this resistance is most likely driven by amino acid changes in the
mitochondrially encoded cytb, more specifically in the ubiquinol oxidizing pocket of cytb (cytb Qo)
of the mitochondrial bc1 complex. We also screened for potential involvement in bifenazate
resistance of the Rieske iron-sulfur protein (ISP) and cytochrome c1 (cytc1), both nuclearly encoded
components of the bc1 complex, in bifenazate resistant T. urticae strains harbouring different cytb
genotypes.
Fascinating aspects about bifenazate resistance evolution in spider mites both in laboratory and field
are the extremely rapid emergence speed and the link with cytb amino acid changes. However, very
little is known about the processes driving the development of this putative mitochondrially
encoded target site resistance. In Chapter 3 we describe a low resistant T. urticae strain harbouring
heteroplasmic mites showing how mtDNA inheritance and the presence of a mitochondrial
bottleneck plays a role in the development of resistance. The influence of random drift on changes
in mutation frequency was assessed by examining the relationship between mutation frequency and

Scope and thesis outline ····························································································
III.
expression of a bifenazate resistance phenotype. We also tried to assess a mutation frequency
threshold in individual mites for surviving field application rate of bifenazate.
Assuming bifenazate acts by inhibition of the mitochondrial cytb Qo, it is likely that the known
bifenazate resistance amino acid changes might confer cross-resistance to other registered acaricidal
QoIs acequinocyl and fluacrypyrim. Chapter 4 shows cross-resistance patterns to fluacrypyrim and
acequinocyl in several strains of T. urticae and P. citri with wild-type and mutated cytb genotypes,
followed by crossing and biochemical assays to attribute the observed resistance to a certain
mitochondrial genotype. We also present a current status of QoI field resistance T. urticae
populations collected from Belgium and the Netherlands. Some cases of bifenazate resistance
putatively not linked with mutated cytb Qo in T. urticae collected from Australia and T. pacificus
collected from California are presented.
In an attempt to further clarify the source of resistance to bifenazate and its mode of action, we
treated T. urticae mites and Leptinotarsa decemlineata beetles with [14
C]bifenazate and performed
biochemical assays in order to show interaction of bifenazate or its metabolites with the
mitochondrial complex bc1 as well as differential metabolism of bifenazate in mites and insects
possibly explaining bifenazate’s high selectivity (Chapter 5). We also investigated whether
mutations or expression levels in the genes encoding for the post-synaptic inhibitory GABA
receptors in the nervous system could be correlated with bifenazate resistance, given bifenazate was
upon its release presented as a neurotoxic acaricide with a unique mode of action on the GABA
receptors.We summarized all available evidence on the mitochondrial versus the nuclear mode of
action hypotheses.

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Chapter I. Introduction to spider mite control

············································································Introduction to spider mite control
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1. Tetranychidae (spider mites)
1.1 Classification, biology and agricultural relevance
Domain Eukaryota
Regnum Animalia (Metazoa)
Phylum Arthropoda
Subphylum Chelicerata
Classis Arachnida
Subclassis Acari
Superordo Acariformes
Ordo Trombidiformes
Subordo Actinedida (Prostigmata)
Superfamilia Tetranychoidea
Familia Tetranychidae
Subfamilia Tetranychinae
Tribus Tetranychini
Genera Tetranychus, Panonychus
(classification according to Bolland et al. 1998; Lindquist et al. 2009)
Mites and ticks (subclass Acari or Acarina) belong to the class of the Arachnida, together with e.g.
spiders (order Araneae) and scorpions (order Scorpiones). This subclass can empirically be divided
into 2 large groups: ticks (suborder Metastigmata) and mites (e.g. suborders Mesostigmata,
Prostigmata, Astigmata and Cryptostigmata). Mites can be discriminated from other arthropods
based on morphological features. One important feature is their size, since most mites are not taller
than 1 mm (Walter and Proctor 1999). Mites (as well as ticks) have reduced all body segmentation
to 1 complex, which can be artificially divided in the part carrying mouthparts (gnathosoma), the
part carrying the legs (podosoma) and the rest (opisthosoma). Nymphal and adult stages carry 4
pairs of legs, while the larval stage only has 3 pairs. Gall- and rust-mites only carry 2 pairs of legs
in all stages. Wings never occur. Contrary to most mites, many species of the Prostigmata, among
them spider mites, possess eye spots, but they only sense direction and intensity of light. Mites
basically explore their habitats using tactile and olfactory senses (Sato et al. 2003).
Although most mites are parasitic or detritivorous, over 6000 species of phytophagous (plant
feeding) mites are known worldwide. The majority of phytophagous species belong to the obligate

Chapter 1 ···············································································································
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plant feeding superfamilies Eriophyoidea (e.g. gall mites, erinose mites, bud mites, rust mites
(Eriophyidae and Phytoptidae) and Tetranychoidea (e.g. spider mites (Tetranychidae) and false
spider mites (Tenuipalpidae). Only a small number of phytophagous species belong to other
lineages (e.g. Eupodoidea, tarsonemid mites, and single oribatid and acarid mites). The
Tetranychidae family contains 2 subfamilies: Bryobiinae and Tetranychinae. The latter subfamily is
divided into 3 tribes: Tenuipalpoidini, Eurytetranychini and Tetranychini. Members of this latter
tribe are the main species subjected to research and discussion in this thesis and are referred to as
“spider mites” hereafter, in analogy with the suggestion made by Saito (2010).
Phytophagous mites form an integral and important part of the natural ecosystem. Some species,
especially eriophyoid mites, can be utilised for the biological control of weeds, as they keep
selectively plant tissues alive. Contrary to eriophyoid mites, members of the Tetranychidae family
are continuously in search of new plants as soon as their original host has perished due to their
feeding. They use their cheliceral stylets to puncture plant cells and suck up the contents, including
the chlorophyll which causes foliage to become necrotic. Spider mite attacks result in significant
losses in a wide range of field and greenhouse crops (Jeppson et al. 1975; Evans, 1992; Walter et al.
2009). The tetranychid family includes over 1200 species, including a number of species feeding on
economically important plants (Zhang 2003; Vacante 2010; Saito 2010). Tetranychid mites owe
their common name, i.e. spider mites, to the silken threads they produce, which assist them in
movement and attachment (Saito 1977; Walter and Proctor 1999). This silk webbing permits a
relatively easy migration through crops by means of leaf to leaf walking on silken bridges and by
using them to stay attached to the plant when dropped from leaves by wind or convection currents.
The webbing may also provide mite colonies (relatively low) protection against natural enemies,
adverse climatic conditions and acaricide treatments (Saito 1995).
Spider mites have in general extremely short life cycles: generation times are usually short (10-14
days) resulting rapidly in severely damaging populations. There are 5 life stages: egg, larva,
protonymph, deutonymph and adult. The larval and nymphal stages are ended by an inactive phase,
called the proto-, deuto- and teleiochrysalis (Figure 1.1).

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Figure 1.1 Schematic representation of the life cycle of spider mites:
a) egg
b) larva
c) protochrysalis
d) protonymph
e) deutochrysalis
f) deutonymph
g) teleiochrysalis
h) adult female
i) adult male
During the chrysalis or resting phase the larva/nymph attaches to a leaf or web and prepares a new
cuticle before ecdysis. Adult males hatch before female adults. Each male then searches for and
guards a female teleiochrysalis. Immediately after female emergence, copulation takes place
(Crooker 1985). Interestingly, most spider mites show arrhenotokous reproduction: females develop
from fertilised eggs and thus are diploid (i.e. owning two sets of chromosomes), whereas males
develop from unfertilized eggs, and are haploid. This haplodiploidy feature has important
consequences for resistance evolvement en evolution (see next section).
Mites belonging to the genus Tetranychus are polyphagous, feeding on a wide variety of plants
(Gutierrez and Helle 1985). On the other hand, the genus Panonychus is considered oligophagous.
Spider mites use the silken threads they produce in different ways to survive on the leaf surface of
their host plants. This finding permitted Saito (2010) to define “life type” as the strategy by which
mites use silken threads on host plant leaves, providing an additional criterion to discriminate
between different species. Tetranychus species produce the largest and most complex webbings and
use them for shelter and transport, while Panonychus mites are known for their less dense
entanglements mainly used to cover the eggs. Table 1.1 summarises briefly some biological features

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of the 4 economically important spider mite species used in this study. The developmental rate is
mainly temperature driven and proceeds faster at higher temperatures, but also relative humidity is
an important factor. In optimal conditions (temperature = 25°C; RH = max. 70 %), development for
these species is completed in 10 days. If temperature gets lower than 10 °C or higher than 35 °C
development stagnates. Temperatures higher than 40 °C kill all stages. It is noteworthy that for
intra- and inter-specific comparison of life history studies, there is an important complication. Most
of the life history studies have been conducted using the leaf disc method (also called detached leaf
culture) to maintain the mite colonies at certain temperatures in laboratory. However, because of
water evaporation from the cotton and Petri dishes used in these assays, temperature decreases
significantly. Saito and Suzuki (1987) showed that the surface temperature of a detached leaf is
significantly lower than the air temperature depending on the air humidity (RH). As a consequence,
without knowing the exact air humidity as it is the case in many life history studies for spider mites,
it is not possible to determine the accurate developmental rates. Shortened daylength together with
decreased temperatures and insufficient food sources induce diapause, a neurohormone mediated
dynamic form to overwinter characterized by low metabolic activity (e.g. no feeding) and reduced
morphogenesis (e.g. no reproduction).

························································································································································ Introduction to spider mite control
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Table 1.1 Comparison of biology variables between 4 spider mite species used in this thesis (Jeppson et al. 1975; Bolland et al. 1998; Bounfour and Tanigoshi
2001; Saito 2010). rm = intrinsic rate of increase, Ro = net reproductive rate, λ = e^rm =finite rate of increase.
Tetranychus urticae Koch Tetranychus pacificus
McGregor
Panonychus citri
McGregor
Panonychus ulmi Koch
Common name(s) two-spotted spider mite, greenhouse
spider mite, red spider mite
Pacific (spider) mite citrus red (spider)
mite
European red (spider)
mite, fruit tree spider
mite
Host interaction specificity low low high high
main problem polyphagous: numerous crops, orchards
and ornamental plants, in field en
greenhouse
cotton, almond, Vitis
spp.
citrus trees fruit crops (apple, pear,
abricot, peach, Prunus
spp., Ribes spp.)
occasional problem - Melia azedarach,
Philadelphus
gordonianus, Ribes spp.,
Vicia spp..
pear, grape, almond,
castor bean,
evergreen shrubs,
ornamental plants
(rose)
ornamental plants (rose)
Life typea
CWa
: always pulling out threads when
walking/aggregating in leaf depressions,
forming web structures. Mites tend to
walk, to deposit eggs and feces, and to
remain quiescent within accumulated
webs.
CWa
: always pulling out
threads when
walking/aggregating in
leaf depressions, forming
web structures. Mites
tend to walk, to deposit
eggs and feces, and to
remain quiescent within
webs.
LWa
: pulling out
threads through the
pedipalpi and making
“guy ropes”
extending from eggs
to leaf surface
LWa
: pulling threads
through the pedipalpi
and making “guy ropes”
extending from eggs to
leaf surface
Life cycle and life
table parametersd
rm (day-1
) 0.218-0.282 0.207-0.290 0.162-0.171 0.180-0.185
Ro (-) 65-74.8 44-108.3 24.4-28.3 -
λ (-) 1.243-1.326 1.229-1.340 1.176-1.186 1.197-1.203
fecundity (eggs/female)b
86.4 – 99.2 (= + 10 eggs/day) max. 50 max. 25 (= 2.5
eggs/day)
max. 45 (= 4.5
eggs/day)
temperature (°C) / RH
optimum (%)
24-29 °C/ < 50% 12 °C threshold 25 °C/ 65 % 23 – 25 °C/50-70 %
in field massive adults in spring-summer-
autumn
massive adults in host
growing season
2 adult number peaks
(spring and winter)
adults most abundant in
summer

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Table 1.1 continued
Tetranychus urticae Koch Tetranychus pacificus
McGregor
Panonychus citri
McGregor
Panonychus ulmi Koch
Life cycle and life
table parametersd
in greenhouses no diapause (long photoperiods, high
temperatures and sufficient food
sources)
no diapause (long
photoperiods, high
temperatures and
sufficient food sources)
no diapause (long
photoperiods, high
temperatures and
sufficient food
sources)
no diapause (long
photoperiods, high
temperatures and
sufficient food sources)
diapause form adult females (orange, without
idiosomac
)
adult females no diapause form winter eggs
Appearance of
development stages
egg spherical, from glassy in the beginning
to hazily yellowish with black point on
top before hatching
spherical, from glassy in
the beginning to hazily
before hatching
hazily red, almost
spherical (flattened at
the base); small shaft
with hairs reaching
the leaves
spherical, stonered, with
small shaft/winter eggs:
intensely red; laid on
sheltered places /
summer eggs: varying
colours, less pigmented
and smaller proportions;
laid on leaves
larva first transparent, green-brown and
idiosoma after feeding
first transparent, green-
brown and idiosoma
after feeding
deeply red to purple red or bright orange
protonymph light-dark green light-dark green deeply red to purple fist lemon yellow, later
orange-red like adults
deutonymph bigger than protonymph, sex
differentiation
bigger than protonymph,
sex differentiation
bigger than
protonymph, sex
differentiation
bigger than protonymph,
sex differentiation
adult orange, yellow, green, brown or black orange, yellow, without
idiosoma
deeply red to purple,
solid dorsal hairs
from red bumps
red or brown, solid
dorsal hairs from more
than 8 white bumps
a
CW = nonsystematic and complex web type; LW = little web type according to Saito 2010; b
considered for 25 °C and RH of 50-70 %; c
idiosoma = two dark feeding spots as a result of food
material in the intestines on either side of the body; d
according to references mentioned in Helle and Overmeer (1985).

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In the middle of the last century, mites moved into the focus of attention as pests relevant to
agriculture, forestry and landscape horticulture, presumably in direct reaction to the “green
revolution” that involved plant cultivation in large-plot monocropping systems, improved methods
of cultivation, selection of high-yielding cultivars and intensified use of pesticides and mineral
fertilizers.
Agroecosystems in which phytophagous mites have become harmful organisms are primarily
orchards, vineyards, greenhouses, urban greeneries, plant nurseries and stored plant products, as
well as, to a somewhat lesser degree, annual field crops. T. urticae is the most important
polyphagous species, with over 1000 different host species and causing enormous yield losses
worldwide in field and greenhouse crops. They are a main pest of Solanaceae, Curcubitaceae,
greenhouse ornamentals, annual and perennial field crops (Jeppson et al. 1975; Zhang 2003;
Migeon and Dorkeld 2010). In heavy infestations, crop losses up to 60 % are reported (AGRIS,
1997; Carbonnelle et al. 2007). T. pacificus is mainly known as a pest in cotton, almond and grapes.
In vineyards, T. pacificus infestations cause serious damage through reduction of sugar content
(Welter et al. 1989; Flaherty et al. 1992). P. ulmi is a pest of Rosaceae (apple, pears and plums)
mainly, and also of peach, cherry, cherry plum and almonds to a lesser degree. Infested trees
decrease their annual growth and bud formation for the next year. Yield losses can reach up to 65%.
P. citri is the most serious citrus pest of California, South Africa, Mediterranean area and Japan.
Injury to citrus leaves is most severe when a combination of high mite infestation and dry weather
conditions exists resulting in heavy leaf and fruit drop, twig dieback and even death of large limbs
(Jeppson et al. 1975).
1.2 Integrated control of spider mites
Integrated control of mites can be defined as part of a broader program of integrated management of
populations of harmful organisms (i.e. integrated pest management or IPM), which implies
combination and harmonisation of more preventive and/or curative control tactics of the key pest in
an agroecosystem. It emphasizes a more thorough study of the ecology of pests, the greater use of
biological control agents, a wider array of other non-chemical tactics of pest control, and better chemical
pesticide management (van Lenteren and Woets 1988; Croft 1990; Blommers 1994; Albajes et al.
1999; Agnello et al. 2003, Beers et al. 2005). According to the IPM guidelines, the control of spider
mites nowadays combines adapted cultivation measures (i.e. agricultural practices and physical
measures), biological control and chemical treatments and is broadely accepted as a sustainable

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model of plant protection (Freier and Boller 2009). Importantly, sustainable integration of
biological and chemical measures essentially needs significant studying of effects of pesticides
(acaricides, insecticides and fungicides) on phytoseiids and other predators, in order to identify
physiological and/or operational selective active substances. Predators come in contact with
pesticides if they are directly treated, exposed to residues or when eating contaminated prey or
pollen of systemically treated plants. In addition to lethal effects (mortality), various pesticides
cause sublethal effects, affecting biological parameters and/or behaviour of survivors (Blümel et al.
1999; Desneux et al. 2007).
1.2.1 Cultivation measures to control spider mites
Cultivation may be adapted in order to prevent spider mite infestations. More resistant cultivars of
crop plants may be used. For example, cultivation of resistant varieties of fruiters and whitewashing
of tree trunks are important preventive control measures against P. ulmi. In future, transgenic plants
resistant to spider mite infestation may provide additional protection of crops. In greenhouses, good
results are obtained with controlled atmosphere treatment. Higher concentrations of nitrogen or
carbon dioxide (Held et al. 2001), as well as increasing the relative humidity to a foggy level (RH >
90%) can significantly reduce the population of spider mites (Honey et al. 2004), however, one has
to consider cultivation guidelines for each crop invidually.
1.2.2 Biological and physical control of spider mites
Biological control is mainly based on the use of natural enemies. For T. urticae, phytoseiid mites
(Phytoseiulus persimilis, Amblyseius californicus, Typhlodromus occidentalis), predatory bugs
(Orius spp., Anthocoris spp.), the predatory gall midge Feltiella acarisuga (former name:
Therodiplosis persicae) and the ladybeetle Stethorus punctillum are successfully used, mainly in
greenhouses. The phytoseiids Phytoseiulus persimilis, P. longipedi, Amblyseius occidentalis and
Typhlodromus occidentalis are the main natural enemies used for control of T. pacificus in
greenhouse and field (Stavrinides and Mills 2011). P. ulmi population density in orchards depends
on the activity of predators, especially phytoseiids belonging to the genus Typhlodromus (T. pyri, T.
occidentalis) spp. Other known natural enemies are Anthocoris nemorum, the predatory mirid bugs
Blepharidopterus angulatus and Sejanus albisignata, Stethorus spp., the lacewing Chrysopa
carnea, Amblyseius subsolidus, Paraseiulus incognitus, the gall midge Arthrocnodax spp. and the
predatory trips Xylapothrips spp. (Collyer 1976). P. citri colonies are biologically controlled mainly

-10-
by members of the genus Amblyseius (A. degenerans, A. californicus) (Zhang 2003; Sosnowska and
Fiedler 2009). Other studies showed that entomopathogenic fungi, especially ascomycetes, may be
useful to control harmful arthropod populations, whether they are used in conventional biological
control applied as classical biological control agents (Hajek et al. 2007; Hajek and Delalibera 2010),
or used formulated as commercial myco-acaricides/insecticides (Maniania et al. 2008, Jackson et al.
2010). Important fungal pathogens of spider mites are Beauveria bassiana, Hirsutella thompsonii,
Lecanicillium spp., Metharizium anisopliae, Isaria fumosorosea, Neozygites floridana (Chandler et
al. 2000; Maniania et al. 2008), of which the conidia and blastospores are used for commercial
preparations. As fungal infection requires high relative humidity, myco-acaricides might be more
effective in protected areas than in the open field (Maniania et al. 2008). Application of predators
and other biological agents proved to be a successful alternative to conventional chemical control of
phytophagous mites, especially when it comes to crops in greenhouses (Albajes et al. 1999; Gerson
and Weintraub 2007; Pilkington et al. 2007). Besides the benefits it possesses, however, biological
control is faced with significant limitations, among them the lack of effective natural enemies for
some spider mites, short vegetation culture that does not leave enough time for successful
implementation of biological control measures, zero tolerance for harmful organisms (e.g.
ornamental plants, especially for T. urticae), too rapid development of spider mites in suitable hosts,
the impact of environmental factors (e.g. too hot and dry climate limits the application of P.
persimilis), the risk of accompanying the introduction of exotic species (van Lenteren and Woets
1988; Gerson et al. 2003). Therefore, the application of acaricides, i.e. synthesised chemical
compounds used to control acarines, is often still necessary.
A physical control method, application of oils in plant protection, is one of the most important and
most natural methods of pest control (Vincent et al. 2003). Highly refined mineral oil can be applied
during the growing season to combat spider mites, like T. urticae on roses in the greenhouse
(Ničetić et al. 2001), or P. ulmi in apple orchards (Eslick et al. 2002; Fernandez et al. 2005). It is a
product of relatively favourable toxicological and ecotoxicological properties, whose mode of
action – preventing the mites of physical gas exchange through blocking the respiratory system of
the mites causing death by suffocation – virtually eliminates the risk of developing resistance
(Agnello et al. 1994; Eslick et al. 2002). In addition to mineral oils, vegetable oil emulsions, e.g.
soybean oil (Lancaster et al. 2002; Moran et al. 2003) or rapeseed oil (Kiss et al. 1996; Marcic et al.
2009), proved to be effective as well.

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1.2.3 Chemical control of spider mites
Although there are some natural products with acaricidal activity (e.g. azadirachtin, spinosad,
myco-acaricides) gaining importance, the use of synthetic acaricides in chemical control is still the
major control strategy, especially in unprotected field crops where biological control has not been
adopted yet and for correcting biological control failures in protected crops (Zhang 2003; Vacante
2010). For instance, growers of grape in Californian vineyards treat around 100,000 ha with
acaricides annually at a cost of 20 million dollars, mainly to control spider mites (Anonymous
2007). Acaricides belong to different chemical groups, with different modes of action (Table 1.2).
The first known acaricides were of natural origin, like sulfur, lime sulfur and petroleum oils. From
1930 on, synthetic acaricides were developed, commercialised and applied on large scale (Vacante
2010). Similar to insecticides sensu stricto and antihelmintics, the majority of acaricides are
neurotoxins targeting AChE, the voltage-gated nerve membrane sodium channel or ligand-gated
nerve membrane chloride channels (GABA-gated chloride channel, glutamate-gated chloride
channel, nACh-gated chloride channel), such as organophosphates, carbamates, pyrethroids,
cyclodiene organochlorines, phenylpyrazoles, avermectins, milbemycins and spinosyns (Lees and
Bowman 2007). Owing to ecotoxicological considerations, a signifcant number of neurotoxic
acaricides have been withdrawn from the market due to more stringent legislations, e.g. the
European Council directive 91/414/EEC (Table 1.2; EU 2010). In the search for more specific and
selective pesticides, several new and structurally diverse acaricides inhibiting mitochondrial
respiration, growth and development have been synthesised and commercialised in the last twenty
years. Acaricides targeting mitochondrial respiration belong to a wide range of chemistries:
pyridazines, pyrazoles, quinazolines, naphthoquinones, β-methoxyacrylates, pyrroles, thioureas and
pyrimidines. Compounds affecting growth and development belong to other chemical groups, like
benzoylureas, tetrazines, tetronic acids and oxazolines (Dekeyser 2005, Lees and Bowman 2007).
Based on the biological mode of action, acaricides can be divided into three classes: chemicals that
are primarily toxic to eggs (ovicides), those showing high toxicity to larvae (larvicides), and others
having toxicity to all motile stages.

-12-
Table 1.2 Acaricides and insecto-acaricides listed by target site and mode of action following IRAC
classification (IRAC 2011; Petanovic et al. 2010a,b; Fytoweb 2011).
Target site/Mode of action Acaricide (a.i.) Annex I1)
Belgium2)
Nervous system
Inhibitors of AChE OP3)
+ +
CARB4)
+ -
Modulators voltage gated Na-channel pyrethroids5)
+ +
Antagonists of GABA-gated chloride channel endosulfan - -
Activators of glutamate-gated chloride channel abamectin + +
milbemectin + +
Allosteric activators of nACh-gated chloride channel spinosad + +
Agonists of octopamine receptors amitraz - -
Growth and development
Inhibitors of mite growth clofentezine + +
hexythiazox + +
Inhibitors of chitin biosynthesis benzoylureas6)
+ +
etoxazole7)
+ +
Inhibitors of ACCase spirodiclofen + +
spiromesifen (+) (+)
spirotetramat + +
Mitochondrial respiration
Inhibitors of complex I pyridaben + +
tebufenpyrad + +
fenpyroximate + +
fenazaquin - -
Inhibitors of complex II cyenopyrafen -* -
Inhibitors of complex III = QoIs acequinoyl (+) -
fluacrypyrim -* -
hydramethylnon - -
bifenazate8)
+ +
Inhibitors of complex V ATP synthase fenbutatin oxide + +
cyhexatin - -
azocyclotin - -
propargite - -
diafenthiuron - -
tetradifon - -
Uncouplers of oxidative phosphorylation chlorfenapyr - -
Unknown mode of action
azadirachtin -** -
benzoximate - -
dicofol - -
chinomethionat - -
1)
Annex I = status under the European Union Directive 91/414 (EU, 2010), + included; (+) pending/in
process; - not included
2)
Belgium = registration status in Belgium, + registered; - not registered
* not registered in EU (European Union)
** withdrawn from the market, applications for inclusion in Annex I re-submitted
3)
dimethoate, chlorpyrifos, chlorpyrifos-methyl
4)
formetanate
5)
cypermethrin, β-cyfluthrin

Chapter 1 ···············································································································
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6)
diflubenzuron, teflubenzuron, lufenuron; lufeneron not registered in Belgium
7)
inhibition of chitin biosynthesis (Nauen and Smagghe 2006)
8)
inhibition of mitochondrial cytb Qo (Van Leeuwen et al. 2008; Van Nieuwenhuyse et al. 2009; this thesis)
2. Mitochondria as targets of acaricides
Mitochondria occupy a central position in the biology and physiology of cells. Most eukaryotic
cells contain many mitochondria (hundreds to thousands per somatic cell, up to 100,000 in
mammalian oocytes), which contain many copies of mtDNA molecules (up to thousands per
mitochondrion). Collectively, they can occupy as much as 30% of the volume of the cytoplasm.
Hundreds of proteins in these crucial organelles are involved in numerous biosynthetic and
degradative reactions fundamental to cell function, including the oxidative phosphorylation
(OXPHOS) pathway yielding the fundamental energy form ATP. Their activities depend on the
distinctive mitochondrial structure, with the different proteins localised in transmembrane position,
at the inner or outer face of the inner or outer membrane, or in the aqueous compartments (matrix).
2.1 Mitochondrial electron transport (MET)
2.1.1 Oxidative phosphorylation (OXPHOS) chain
Biological energy conversion is mainly conducted in membranes such as the mitochondrial inner
membrane in eukaryotic cells and the plasma membrane in bacteria. Here, chemical energy is
converted into a proton gradient and a membrane potential that is then used in various cellular
processes, including the synthesis of adenosine triphosphate (ATP). In eukaryotes, this oxidative
phosphorylation (OXPHOS) process is procured in 5 enzyme complexes, each with its specific
function (Figure 1.2). Complex I (NADH dehydrogenase, NADH:ubiquinone oxidoreductase) is the
largest membrane bound enzyme complex transferring protons from the mitochondrial matrix to the
intermembrane space (IMS). It forms, together with complex II, the start of the mitochondrial
electron transport (MET). Complex II (succinate dehydrogenase, succinate:ubiquinone
oxidoreductase), which is part of the Krebs cycle, is the only enzyme complex that does not transfer
protons to the intermembrane space (IMS). Complex III (ubiquinol:cytochrome c oxidoreductase,
QCR, bc1) is most extensively studied enzyme complex and known for its Q cycle (see 2.1.3). The
complex IV (cytochrome c oxidase, COX) is the final station of the MET where oxygen is reduced
to water. Complex V (ATP phosphohydrolase, ATP synthase, FoF1 H+-ATPase) catalyses the
formation of ATP through the proton gradient created by the complexes I, III and IV. Interestingly,

-14-
these enzyme complexes may form supramolecular complexes (supercomplexes or respirasomes),
as they are not floating independently from each other on the inner membrane (Schägger and
Pfeiffer 2000; Wittig et al. 2007a,b; Yan and Forster 2009; Juszczuk and Rychter 2009).

························································································································································ Introduction to spider mite control
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Figure 1.2 Schematic representation of different OXPHOS complexes of the mitochondrial electron transport chain in eukaryotes and their functions (redrafted from
Kurosu et al. 2010). Mitochondria oxidise metabolic substrates (= the electron donors), recovered from carbohydrates and fats in the the tricarboxylic acid cycle (or Krebs cycle)
and through β-oxidation respectively, in order to produce H2O and ATP, with O2 acting as the terminal electron acceptor for the electron transport chain that generates the proton
gradient across the inner mitochondrial membrane. The resulting electrons are transferred to the MET chains via Complex I (NADH dehydrogenase, NADH:ubiquinone
oxidoreductase) or Complex II (succinate dehydrogenase, succinate:ubiquinone oxidoreductase) and then flow to ubiquinone (coenzyme Q) which is reduced to ubiquinol (QH2).
Subsequently, QH2 is transformed in the Q cycle (complex III, ubiquinol:cytochrome c oxidoreductase, QCR, bc1) to give ubisemiquinone (SQ) and Q, transferring electrons to
the mobile electron carrier cytochrome c and eventually to cytochrome c oxidase (complex IV, COX). Finally in complex IV, four one-electron transfers to oxygen (1/2 O2) occur
resulting in the formation of H2O, representing the endpoint of the MET. The energy released by the electron transport chain is used to pump protons out of the inner
mitochondrial membrane, creating the transmembrane proton gradient or protonmotive force. As a result, the potential energy stored in this proton gradient is used to condense
adenosine diphosphosphate (ADP) and a phosphate group Pi to make ATP via complex V (ATP phosphohydrolase, ATP synthase, FoF1 H+
-ATPase), driven by the movement of
protons back through a complex V proton channel. Matrix ATP is then exchanged for cytosolic ADP by the adenine nucleotide translocator.

-16-
2.1.2 Mitochondrial genomes
Notably, the OXPHOS enzyme complexes, with the exception of complex II, consist of both
mitochondrially and nuclearly encoded polypeptides, which are intricately linked to enable the
enzymatic reactions. Proteins from mitochondrial origin are encoded by the mtDNA and are
assembled within the mitochondria. With a few exceptions, the typical animal mitochondrial gene
complement contains 37 genes: 13 genes encoding protein subunits of complex I, III, IV and V and
24 genes encoding the translational machinery of the mtDNA itself necessary for the translation of
these mitochondrially encoded proteins (2 rRNAs and 22 tRNAs) (Table 1.3). The other proteins,
encoded by nuclear DNA (nDNA), are synthesised in the cytosol and subsequently imported into
mitochondria through protein translocation machineries of the outer and inner membranes (Boore
1999; Stojanovski et al. 2003).
Table 1.3 Genes encoding 13 OXPHOS complex subunits, 2 rRNAs and 22 tRNAs in typical
animal mitochondrial genome (adapted from Boore 1999)
Gene (Synonym) Polypeptide encoded site of function
ND1,2,3,4,5,6,4L
(nad1,2,3,4,5,6,4L)
NADH:ubiquinone oxidoreductase subunits 1,2,3,4,5,6,4L OXPHOS complex I
cytb (cob) cytochrome b apoenzyme OXPHOS complex III
COI,II,III
(cox1,cox2,cox3)
cytochrome c oxidase subunits COX1,2,3 OXPHOS complex IV
ATP6,8 (atp6,8) FoF1 H+
-ATPase subunits 6,8 OXPHOS complex V
srRNA (rrnS) 12S ribosomal subunit RNA mitochondrial matrix
lrRNA (rrnL) 16S ribosomal subunit RNA mitochondrial matrix
trnX1)
18 tRNAs each specifying a single amino acid mitochondrial matrix
L(CUN), L(UUR) 2 tRNAs specifying amino acid L mitochondrial matrix
S(AGN), S(UCN) 2 tRNAs specifying amino acid S mitochondrial matrix
1)
X = corresponding single letter amino acid code
2.1.2.1 Biology of mitochondrial genomes and mutagenesis
Apart from the cell nucleus, mitochondria are the only animal cell organelles containing DNA.
Until recently it was generally accepted that the eukaryotic cell is the product of an endosymbiosis
between a primitive eukaryotic cell and an α-Protobacterium (Rickettsia sp.) according to the “serial
endosymbiont hypothesis” (Margulis 1981). During evolution an off-loading of the bacterial genes
to the host genome occurred forming the nuclear genome and an early stage mitochondrial genome
evolved from the remaining bacterial genes (Andersson et al. 1998). However, with the discovery of
hydrogenosomes, i.e. cell organelles containing mitochondrial proteins, producing H2 as a by-
product of ATP synthesis, a new concept of the origin of mitochondria has been postulated which is
not linked with a symbiosis based on oxidative phosphorylation, but with a symbiosis based on

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hydrogen metabolism. The “host” is now supposed to have been a hydrogen requiring
Archaebacterium and the “metabolic symbiont” a hydrogen producing eubacterial species.
Subsequently the eubacterial genome made a major contribution to the nucleus, and the remnants of
the eubacterial genome then developed into mitochondrial DNA as we know it today. This
“hydrogen hypothesis” may cause a drastic revision of the opinion that mitochondria are the direct
descendants of a bacterial endosymbiont that became established at an early stage in a nucleus-
containing (but non-mitochondrial) host cell. Instead, the origin of mitochondria is placed very
close to, if not coincident with, the origin of the eukaryotic cell itself. Moreover, there might be an
ongoing process of transfer of mitochondrial genes to the nucleus and at the “evolutionary end” all
mitochondrial genes might be found in the nucleus (Gray et al. 1999; de Coo 2005).
One mitochondrion harbours numerous copies of mtDNA molecules (up to 1000 copies in human
somatic cells), which are in most species circular (Nosek et al. 1998), and RNA molecules (tRNA,
rRNA). MtDNA is a “small” molecule with a unique biology. The mitochondrial genomes differ
from nuclear genomes in many ways, such as size (e.g. human mitochondrial genome is only
0.00055 % of the total human genome), mutation rate, repair mechanism, mode of inheritance,
degree of recombination, number of introns and ploidy (Scheffler 2001). The smallest
mitochondrial genome known is that from the human malaria parasite Plasmodium falciparum
which consists of less than 6,000 base pairs (bp) coding for 3 genes. The largest mtDNA known is
that from the plant Arabidopsis thaliana consisting of 366.9 kbp coding for 32 genes. The
mitochondrial DNA from the protozoan Reclinomonas americana is the most gene-rich to date,
harbouring 97 genes.
Mitochondria are the powerhouse of the cell, but due to the large potential difference generated
from the transfer of hydrogen from carbon (originally in carbohydrates and fats) to oxygen (H2O)
the MET chain might be short circuited resulting in incomplete processing of oxygen and/or release
of free electrons. As a consequence, oxygen radicals (reactive oxygen species or ROS) are
produced, such as superoxide, hydrogen peroxide and hydroxyl radicals. ROS products can damage
DNA, lipids and proteins, and their production increases with age. Generation of ROS at complexes
I and III is believed to be the major ROS source, with up to 1 - 2% of the oxygen consumed being
converted to ROS (Ballard and Whitlock 2004). Most likely, mtDNA is a primary target of
oxidative damage resulting in point mutations or major deletions eliminating more whole genes
(Scheffler 2001). The lack of histones (which are protective against free radicals), the inefficient
repair mechanisms and the near location of mtDNA to the inner mitochondrial membrane (the
major site of ROS production) promote the oxidative damage to mtDNA nucleotides (Bohr and

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Anson 1999; Kirkinezos and Moraes 2001; Mason et al. 2003; Hashiguchi et al. 2004; Boesch et al.
2011). Moreover, mtDNA has very few non-coding sequences (i.e. introns), therefore increasing the
likelihood of a mtDNA alteration to affect a gene product. As a consequence, mutation rates of
mtDNA are generally higher than those of nDNA. For example, Drosophila mitochondrial genes
have 1.7 - 3.4 times higher synonymous substitution rates (defined as the number of mutants
reaching fixation per generation) than the fastest nuclear genes or 4.5 - 9.0 times higher rates than
the average nuclear genes (Moriyama and Powell 1997; Lynch and Blanchard 1998). As more
copies of the mtDNA sustain mutations, complexes in the MET chains are expected to become less
efficient as a result of stoichiometric mismatches between the mitochondrially and nuclearly
encoded components.
Another conspicuous aspect of most animal mtDNA, contrary to fungal and plant mtDNA, is that it
does not usually undergo genetic recombination, although the biological machinery for
recombination seems to be present in the mitochondria (Kroon et al. 1978; Thyagarajan et al. 1996;
Kajander et al. 2000; Birky 2001). Moreover, the mitochondrial genome is maternally inherited,
although partial biparental inheritance at low frequency, usually from paternal leakage, is known for
Drosophila spp., mice, birds and humans (Kondo et al. 1990; Gyllensten et al. 1991; Schwartz and
Vissing 2002; Kvist et al. 2003). A likely evolutionary explanation for the uniparental transmission
is that it prevents the spread of deleterious mutations in the mitochondrial DNA. As there are only
limited DNA repair mechanisms in mitochondria, a deletion in the mitochondrial DNA may give it
a replication advantage, eventually replacing all mitochondrial DNA in the population. Uniparental
transmission limits the spread of the mutated mtDNA to only one parental line, thus protecting the
population as a whole from extinction. Biases toward A/T-ending codons have also been observed
in the mtDNA of many species. This may be caused by factors that influence mutation rate and the
relative availability of nucleotides in the mitochondrial matrix (Martin 1995; Xia et al. 1996).
Alternatively, it is likely that selection may also be involved in the high A/T content of mtDNA:
A/T-rich genomes may replicate more quickly than G/C-rich genomes and have a selective
advantage in a heteroplasmic population (Denver et al. 2000; Ballard 2000). A final remark is that
to the lack of recombination together with the physical linkage of all sites in the mtDNA molecule
make traditional genetic approaches for mitochondrial genome evolution impossible.

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2.1.2.2 Biological and clinical relevance of mitochondrial genomes
Due to the lack of genetic recombination, their small sizes and high copy numbers, mitochondrial
genomes have been used in the last three decades as a tool for phylogenetic studies of species and
populations (Downton 1999; Rawlings et al. 2001). Gene order and sequence similarities may
predict the evolutionary relationship between different taxa (e.g. Boore and Brown 1998).
Moreover, due to their small size and extreme gene density, complete mitochondrial genome
sequences were recently introduced as a model for exploring genome evolution (Boore et al. 2005;
Dowling et al. 2008). Interestingly, the elucidation of the genetic basis for a large number and
variety of so-called mitochondrial diseases, starting in the late 1980s, has made studies of
mitochondria more relevant to the medical community (Scheffler 2001). Figure 1.3 gives an
overview of mitochondrially encoded human diseases. More than 100 pathogenic mutations are
known in the mtDNA (Servidei 2004). Interestingly, single amino acid substitutions in the
mitochondrially encoded cytb are involved in important human diseases. Missense mutations and
in- and out-frame short deletions have been reported in patients with various diseases, in addition to
a few nonsense mutations (Table 1.4).
Table 1.4 Primate cytb amino acid changes associated with human diseases (redrafted from Fisher
and Meunier 2001; Azevedo et al. 2009). LHON = Leber’s hereditary optich neuropathy. MELAS =
mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes.
Bovine residue Amino acid change Disease Reference(s)
G34 G34S Exercise intolerance Andreu et al. 1998
G166 G166E Cardiomyopathy Valnot et al. 1999
D171 D171N LHON Johns and Neufeld (1991)
L236 L236I Cardiomyopathy Valnot et al. 1999; Marin-Garcia et
al. 1996
G251-P258 deletion of residues G251-
P258
Myopathy Andreu et al. 1998
G251 G251D/G251S Cardiomyopathy/Exer-
cise intolerance
Andreu et al. 2000, Azevedo et al.
2009
G290 G290D Exercise intolerance Dumoulin et al. 1996
G339 G339E Myopathy Andreu et al. 1998
V356 V356M LHON Johns and Neufeld (1991)
- deletion of 4 bp parkinsonism/
MELAS overlap
syndrome
de Coo et al. 1999

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Figure 1.3 Mitochondrial disease phenotypes associated with mutations in mtDNA encoded genes
(redrafted from Shoubridge and Wai 2007). The structural and rRNA genes are shown as shaded bars:
ND1–5, subunits of complex I; Cytb, subunit of complex III; COI,II,III, subunits of complex IV; ATP6,8,
subunits of complex V. The tRNA genes are depicted as solid circles using the single letter amino acid code.
The major clinical phenotypes associated with mutations in the individual genes are shown in boxes next to
the position of the gene. The extent of the common deletion, which is associated with Kearns–Sayre
syndrome and removes five tRNA genes, is indicated by lines inside the genome. CPEO, chronic progressive
external ophthalmoplegia; LS, Leigh syndrome; PECM, progressive encephalomyopathy; MELAS,
mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonus epilepsy
and ragged red fibers; LHON, Leber’s hereditary optic neuropathy; NARP, neuropathy, ataxia, and retinitis
pigmentosa; MILS, maternally inherited Leigh syndrome.

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2.1.2.3 Mitochondrial genomes of insects and (spider) mites
During the last 10 years, arthropod mitochondrial genomes have been extensively sequenced due to
the improvements of genomic technologies and the interest in mitochondrial genome organisation
and evolution (Boore 2006). Recently, a new integrated platform, named the Insect Mitochondrial
Genome Database (IMGD, www.imgd.org; Lee et al. 2009), has been developed to better integrate
available hexapod mitochondrial gene and genome sequences and to provide bioinformatics tools
for efficient data retrieval and utilisation. The IMGD archives the sequences of 255 completely
sequenced mitochondrial genomes, as well as partial sequences of nearly 342,013 mitochondrial
genomes from about 74,896 insect species (IMGD, Accessed May 2012). Like for other animals,
insect mtDNA is in general a small molecule (12.9-19.5 kbp) containing typically 37 encoding
genes. The GC content ranges from 13% to 35% with an average of 25% (Lee et al. 2009).
To date, the complete mitochondrial genome of 26 Acari species has been sequenced (14 spp. and
12 spp. for superorders Parasitiformes and Acariformes, respectively). Most mitochondrial genomes
are about 15 kb circular molecules and also contain the 37 metazoan genes. However, there are
some execeptions: the mitochondrial genome of Leptotrombidium pallidum and Metaseiulus
occidentalis possesses duplicated genes (Shao et al. 2005; Jeyaprakash and Hoy 2007), while the
mtDNA of the oribatid mite Steganacarus magnus lacks 16 tRNA genes (Domes et al. 2008). In
addition, gene rearrangement, reverse base composition, and atypical tRNA genes are frequently
present in Acari, especially in the Acariformes, such as in the mite genera Leptotrombidium (Shao
et al. 2005, 2006), and Dermatophagoides (Dermauw et al. 2009), and also in the spider mites T.
urticae, P. citri and P. ulmi (Van Leeuwen et al. 2008; Yuan et al. 2010). The circular genomes of
the latter 3 spider mites are extremely adenine and thymine rich (A+T = 84.27%, 85.28%, 85.60%
for T. urticae, P. citri and P. ulmi respectively), which is much higher than the average A+T content
of acarid mitochondrial genomes (A+T = 75.34%). In the horseshoe crab Limulus polyphemus,
which is considered to have the representative ground pattern for arthropod mitochondrial genomes
(Staton et al. 1997; Lavrov et al. 2000), the A+T content is 67.57%. Moreover, spider mite
mitochondrial genomes are among the smallest in the arthropod group (13,103 bp, 13,077 bp and
13,115 bp for T. urticae, P. citri and P. ulmi respectively). This is primarily due to significant size
reduction of protein coding genes, a rRNA gene (rrnL) and the putative control region (i.e. A+T
rich non-coding region with a size of 44 bp, 57 bp and 55 bp for T. urticae, P. citri and P. ulmi
respectively) in comparison with other acarines (Figure 1.4). Up till now, only the springtail
Onychiurus orientalis seems to have a smaller mitochondrial genome (12,984 bp; Lee et al. 2009).
The mitochondrial gene order is the same in the 3 spider mite species. They differ from other Acari

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by a series of gene translocations and/or inversions. Only 4 protein encoding genes have the same
relative position compared to the arthropod reference L. polyphemus: cox1, nad2, nad3 and cytb
(Figure 1.4). Interestingly, the latter gene, also called cob, which encodes the cytochrome b
apoenzyme of 353 amino acid residues, has the same size of 1,065 bp in the 3 spider mite species,.
Together with cox1, it shows the lowest nucleotide substitution rate per site and the lowest Ka/Ks
value (= number of nonsynonymous amino acid substitutions per nonsynonymous site/number of
synonymous amino acid substitutions per synonymous site) when compared to other Acari,
suggesting it is one of the slowest evolving mitochondrially encoded proteins (Yuan et al. 2010).
The fact that gene order is conserved within tetranychids, but not in other acarines, suggests that
significant rearrangements have occurred after spider mites diverged from other acarines.
Moreover, unusual tRNA genes seem to be a common feature for spider mites, as highly truncated
tRNA genes lacking either the T- or D-arm were found in mitochondrial genomes of T. urticae, P.
citri and P. ulmi (Yuan et al. 2010). So far, the scarcity of available sequence data has greatly
impeded evolutionary studies in mites and ticks. However, the information available on tetranychid
mitochondrial genomes might be the start for both the elucidation of the molecular evolution of
functional genes such as insecto-acaricide resistance genes, and development of new acaricides
uniquely targeting mitochondrial gene (see next chapters).

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Figure 1.4 Redrafted from Yuan et al. 2010 (Up) Map of the mitochondrial genomes of three spider
mites T. urticae, P. citri and P. ulmi. Genes coded in the J-strand (clockwise) are red or orange coloured.
Genes coded in the N-strand (counterclockwise) are blue or cyan coloured. The D-loop (putative control
region) is given in green. Alternation of colours was applied for clarity. Protein coding and ribosomal genes
are shown with standard abbreviations. Cob = cyt b. Genes for tRNA are abbreviated by a single letter amino
acid code, with S1 = AGN, S2 = UCN, L1 = CUN, and L2 = UUR. Numbers at gene junctions indicate the
length of small non-coding regions (i.e. introns) where negative numbers indicate overlap between genes,
only valuable for P. citri. (Below) Linear representation of gene rearrangements of the T. urticae, P. citri
and P. ulmi mitochondrial genomes compared to the arthropod reference species L. polyphemus. Only
protein coding genes and ribosomal RNA (rRNA) genes are marked, whereas transfer RNA genes (tRNAs)
are not denoted because they are highly complex. Green boxes represent genes with the same relative
position as in the arthropod ground pattern L. polyphemus. Blue colour indicates translocations only, whereas
yellow colour denotes translocations and inversions combined. Horizontal lines combine adjacent genes,
which are probably subject to a joint inversion. All genes are transcribed from left to right except those
underlined to indicate an opposite transcriptional orientation. Genes are not drawn to scale and non-coding
regions are not shown.

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2.1.3 Complex III and the protonmotive Q cycle
During the respiration, ubiquinol (UQH2, QH2) is oxidised to ubiquinone (Q, coenzyme Q) by
complex III (cytochrome bc1 complex , cyt bc1, bc1, ubiquinol:cytochrome c oxidoreductase
enzyme complex, QCR) in eukaryotic mitochondria and many bacteria (e.g. Rhodobacter spp.,
Pseudomonas spp., Rhodospirillum spp., Rhodopseudomonas spp.). The number of subunits in
mitochondrial apoenzymatic bc1 complex ranges from 9 (yeast) to 11 (bovine), but the catalytic
core is constituted by only 3 proteins: cytochrome b (cytb), cytochrome c1 (cytc1) and Rieske iron-
sulfur protein (ISP) (e.g. Kramer et al. 2004; Xia et al. 2007). Like its mitochondrial counterpart,
the bacterial bc1 is a symmetric dimer but contains only the three catalytic subunits per monomer:
cytb, cytc1 and ISP. The overall structure of these three-subunit bc1 is similar to the corresponding
subunits of mitochondrial bc1 but differs in a number of insertions and deletions as found in R.
sphaeroides, R. capsulatus and Paracoccus denitrificans (Esser et al. 2006; Xia et al. 2007). All
proteins but cytb found in mitochondrial bc1 are encoded by the nuclear genome, so cytb is the only
protein being encoded by mtDNA (i.e. the gene cytb or cob) and assembled in the mitochondria.
Cytb possesses 2 b-type hemes bL en bH with relatively lower and higher redox potentials
respectively, and 2 QH2/Q binding sites Qo and Qi. The Q reducing site, Qi or QN, is located near the
negative side of the membrane (the mitochondrial matrix or cytoplasmic side) and is electronically
linked via the hemes bL and bH to the QH2 oxidising site. This latter site, Qo or QP site, is located on
the positive side of the membrane (intermembrane space) at the interface between cyt bL and the
ISP, and acts during normal turnover to oxidise QH2 to Q. Essentially, the bc1 complex acts as a
proton translocator to store energy in a proton motive force by oxidising QH2 in an electron
bifurcation mechanism at Qo and reducing cytc in the so-called Q cycle (Trumpower et al. 1994;
Zhang et al. 1998). There are still unknown aspects of the exact processes in the Q cycle, which is
subject of much debate, but the most recent broadly accepted model is the adapted catalytic switch
model according to Xia et al. (2007) and Cramer et al. (2011) (Figures 1.5 and 1.6). According to
this model, 2 highly conserved cytb segments, the cd1 helix and the PEWY motif adjoining the ef
helix, play a crucial role in the control mechanism for the two electrons from a quinol molecule,
given in four steps (Figure 1.6): (1) in the apoenzyme (i.e. the protein complex without the
coenzyme), ISP is not fixed in its conformation; (2) as the substrate QH2 enters the Qo pocket, it
initially binds to the distal cytb Qo site, which moves the cd1 helix and opens up the ISP binding
crater, allowing the ISP to be fixed in place; (3) one electron from the quinol goes to the ISP and
another electron goes to the bL heme with one proton deposited into the IMS; (4) with the electron
in the bL heme on its way to the bH heme, the ubiquinone Q moves to the proximal cytb Qo site,

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which brings back the cd1 helix and releases the ISP-ED. The freed ISP-ED then swivels over to
cytc1 and reduces it (Xia et al. 2007).
Figure 1.5 Q cycle scheme in the context of human three-dimensional structure (redrafted from Crofts
2004). The three catalytic subunits of bc1 complex are indicated in colour: cytb (cyan), ISP (yellow),
cytc1(blue). The Q cycle depends on two spatially separated binding sites for ubiquinol (QH2) and
ubiquinone (Q), both located in subunit cytb. The key step of the mechanism is the bifurcated route of the
two electrons released upon ubiquinol oxidation at cytb Qo. One electron is transferred into the high potential
chain, the 2Fe-2S cluster Rieske protein (ISP) and cytc1, consequently being delivered to cytc. The second
electron is transferred via the low potential chain, heme bL and heme bH of cytb, to cytb Qi, at which quinone
is reduced to semiquinone (SQ). A complete turnover of the enzyme requires the oxidation of two QH2,
resulting in reduction of the SQ to QH2.

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Figure 1.6 Schematic cartoon representation of mechanism for electron bifurcation at cytb Qo, binding
of QoIs on 2 distinct niches, and motion control of the ISP extrinsic domain according to an adapted
catalytic switch model for Q cycle (redrafted from Xia et al. 2007). The cd1 helix and PEWY motif in
gray represent a native, unbound configuration. The PEWY motif in cyan stands for the configuration with a
bound quinone form (QH2, semiquinol) or cytb Qo inhibitor. The cd1 helix in pink symbolises the
conformation in the presence of a bound quinone form (QH2, semiquinone) or Pf inhibitor occupying the
distal site: the cd1 helix undergoes an outward translation (i.e. towards ISP-ED, “on” position). The cd1
helix in green shows the conformation when a Pm inhibitor is taking the proximal site: the cd1 helix
undergoes an inward translation (i.e. in the direction of bL, “off” position). The redox conditions of hemes bL,
bH, ISP-ED (marked as 2Fe2S) and cytc1 (marked as c1) are also shown: yellow colour stands for the
oxidised form, purple colour indicates reduced form.

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2.2 Acaricides targeting nuclearly encoded proteins of mitochondrial OXPHOS
complexes I, II, IV and V
In the early 1990ies the application of several chemically unrelated compounds has been
successively introduced (fenpyroximate, pyridaben, tebufenpyrad, fenazaquin, pyrimidifen), sharing
the same mechanism of action, i.e. inhibition of respiratory complex I chain in mitochondria
(Hollingworth and Ahammadsahib 1995). These acaricides, also called METI acaricides
(Mitochondrial Electron Transport Inhibitors), quickly became widely used miticides against
tetranychids and eriophyoids, and are now in widespread use globally. Due to rapid knockdown of
all the developmental stages and long-term residual toxicological effect, METIs showed high
efficiency in suppressing populations resistant to organophosphates, pyrethroids and acaricides
from older generations (Dekeyser 2005; Van Leeuwen et al. 2009). Flufenerim, a newly developed
METI acaricide, will soon be ready for the market (Krämer and Schirmer 2007; Lümmen 2007).
There are little compounds affecting respiratory complex II. Cyenopyrafen, a new compound which
is being prepared for the market, proves to inhibit respiratory complex II, but no further details on
its mode of action are available yet (Krämer and Schirmer 2007; Lümmen 2007). There are no
examples of insecto-acaricides acting on complex IV.
Finally, there are quite a lot of examples of acaricides targeting the mitochondrial complex V. DNP
(2,4-dinitrophenol) was the first developed synthetic insecticide. It was widely used between 1930
and 1950, but due to severe environmental contamination it was withdrawn from the market.
Together with the insecto-acaricide chlorfenapyr, which is still used at present, it is an uncoupler of
oxidative phosphorylation: the production of ATP by the complex V enzyme ATP synthase is
disconnected from oxidation, and thus “uncoupled”. In fact, DNP and the active metabolite of
chlorfenapyr act as protonophores, allowing protons to leak across the inner mitochondrial
membrane and thus bypass ATP synthase. This renders ATP energy production inefficient: part of
the energy that is normally produced from cellular respiration is wasted as heat energy.
Interestingly, chlorfenapyr, derived from halogenated pyrroles, is a pro-pesticide which needs to be
metabolised into an active pesticide after entering the target host. Oxidative removal of the N-
ethoxymethyl group of chlorfenapyr by MFOs (mixed function oxidases) results in the N-
dealkylated compound [1H-pyrrole-3-carbonitrile, 4-bromo-2-(4-chlorophenyl)-5-trifluoromethyl)],
the uncoupler of oxidative phosphorylation in the mitochondria. Chlorfenapyr was introduced as a
broad-spectrum insecticide (lepidopterans, coleopterans, thysanopterans) and acaricide toxic to the
mobile stages of spider mites and eriophyoids. Chlorfenapyr primarily affects the digestive system,
with significant translaminar movements (Dekeyser 2005; Krämer and Schirmer 2007, Van

-28-
Leeuwen et al. 2010). Other acaricides targeting complex V are organotins (fenbutatin oxide,
cyhexatin, azocyclotin) and the widely used propargite, diafenthiuron and tetradifon.
2.3 Chemicals targeting mitochondrially encoded cytb protein of OXPHOS
complex III
2.3.1 Non-insecto-acaricidal inhibitors of mitochondrially encoded cytb
Remarkably, bc1 complexes are highly conserved among different species and show high similarity
with conserved regions of the related b6f complexes in cyanobacteria and chloroplasts as well.
Therefore, the binding site and the inhibitory mechanism of the bc1 inhibitors may be explained on
the basis of the available structural information from yeast, bacterium, rabbit and chicken bc1
apoenzymes and cytb6f apoenzymes in the photosynthetic cyanobacterium Mastigocladus
laminosus and chloroplasts of the alga Chlamydomonas reinhardtii (Xia et al. 1997; Zhang et al.
1998; Iwata et al. 1998; Hunte et al. 2000; Kurisu et al. 2003; Stroebel et al. 2003; Berry et al. 2004;
Esser et al. 2006).
Cytb Qi and cytb Qo form the 2 sites known for inhibitor binding. Known Qi inhibitors are
antimycin, funiculosin, diuron, HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide), NQNO (2-n-
nonyl-4-hydroxyquinoline N-oxide) and azole-fused salicylamides (Brasseur et al. 1996; Bolgunas
et al. 2006). However, cytb Qo forms the major inhibition site with numerous known inhibitors. The
cytb Qo pocket is composed of residues from two sequence segments (Figure 1.7). One segment,
from residue 120 to 182 (CD loop, composed of the helices C, cd1, cd2, and D) (bovine
numbering), another from residue 250-300 (EF loop, containing helices ef and F in eukaryotes, an
additional ef1 helix in some prokaryotes). These 2 regions lie close to each other in the folded
protein and play a role in ligand binding (Esser et al. 2004). X-ray crystallographic research of
inhibitor binding to the cytb Qo pocket revealed a proximal and a distal niche (von Jagow et al.
1986; Link et al. 1993; Crofts et al. 1999). The proximal Qo niche is located close to the bL heme,
while the distal niche is located close to the ISP (Kramer et al. 2004). Most interestingly, the two
niches of cytb Qo bind 2 different classes of cytb Qo inhibitors (QoIs) (Figure 1.7) (Crofts 2004;
Esser et al. 2004, 2006; Xia et al. 2007; Cramer et al. 2011).
The first class, distal niche inhibitors, also called Pf inhibitors (f for fixed ISP) or quinoid inhibitors,
strongly interact with the ISP and bind competitively with QH2 at the distal Qo site where they fix
the extrinsic head carrying the [2Fe-2S] cluster of the ISP (ISP-ED) in the so-called b position
preventing its reoxidation by cyt c1 (Crofts et al. 1999; Lümmen 2007). Known Pf inhibitors are the

Chapter 1 ···············································································································
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2-hydroxynaphthoquinone related compounds stigmatellin, HHDBT (5-heptyl-6-hydroxy-4,7-
dioxobenzothiazol), UHDBT (5-undecyl-6-hydroxy-4,7-dioxobenzothiazol), NQNO (2-n-nonyl-4-
hydroxyquinoline N-oxide), the 2-hydroxynapthoquinones (HONQ) UHNQ (3-undecyl-2-hydroxy-
1,4-naphthoquinone), atovaquone, lapachol and hydrolapachol, and the strobilurin fungicides
famoxadone (5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione) and
JG144 (S-3-anilino-5-methyl-5-(4,6-difluorophenyl)-1,3-oxazolidine-2,4-dione) (Bowyer and
Trumpower 1980; Matsuura et al. 1983; Olliaro and Trigg 1995; Khambay and Jewess 2000; Fisher
and Meunier 2001; Jewess et al. 2002; Palsdottir et al. 2003; Crofts 2004; Esser et al. 2004; Esser et
al. 2006).
The second class, proximal niche inhibitors or Pm inhibitors (m for mobile ISP), bind closer to cyt
bL and do not interact with ISP (Kim et al. 1998; Crofts et al. 1999; Kramer et al. 2004; Xia et al.
2007). Known Pm inhibitors are the β-methoxyacrylate (MOA) type inhibitors strobilurin A
(methyl(E)-3-methoxy-2-(5-phenylpenta-2,4-dienyl)acrylate, synonyme: mucidin), strobilurin B,
azoxystrobin, picoxystrobin, MOA-stilbene (MOAS, 3-methoxy-2-(2-styryl-phenyl)-acrylic acid
methyl ester), and the related strobilurin compounds myxothiazol, oudemansin A and CPMB-oxime
(N-[1,3-bis[(4-chlorophenyl)methoxy]-1,3-diphenylpropan-2-ylidene]hydroxylamine). They all
bind to cytb Qo non-competitively with respect to QH2 substrate (Brandt et al. 1988; Gisi et al.
2002; Crofts 2004; Xia et al. 2007).
Another group of cytb Qo inhibitors, unclassified so far, is composed of the strobilurin fungicides
pyraclostrobin (BAS 500), kresoxim-methyl, trifloxystrobin, metominostrobin (SSF 126),
fenamidone (RPA 407213) and recently developed compounds fluoxastrobin and dimoxystrobin.
Many other strobilurin fungicides are being developed and prepared for the agricultural market, e.g.
enestrobin, orysastrobin and pyribencarb (Gisi et al. 2002; Balba 2007; Liu et al. 2008).

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Figure 1.7 Sequence alignment of the cytb Qo residues (T. urticae numbering) in animals, plants, fungi
and protozoans (inhibitor binding is adapted from Esser et al. 2004; for atavoquone, binding residues
are indicated based on Korsinczky et al. 2000, and Kessl et al. 2003, 2004, 2005). Sequence alignment of
the cytb Qo pocket residues are shown in two sequence segments. The sequences shown include Arabidopsis
thaliana (At, GenBank accession number CAA47966), Bos taurus (Bt, AAZ95350), Capra hircus (Ch,
AAP9735), Drosophila melanogaster (Dm, CAB91062), Equus caballus (Ec, BAA637), Gallus gallus (Gg,
AA044995), Homo sapiens (Hs, AAX15094), Mus musculus (Mm, CAD54444), Oryctolagus cuniculus (Oc,
CAA04859), P. citri (Pc, HM367068), Plasmodium falciparum (Pf, NP_059668), Pneumocystis jeroveci (Pj,
AAC962), Rattus norvegicus (Rn, AAN7760), Saccharomyces cerevisiae (Sc, ABS28693), T. urticae (Tu,
EU345430), Ventura inaequalis (Vi, AAC03553). The secondary structural helices as determined

Chapter 1 ···············································································································
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crystallographically are mapped to the primary sequence as rectangular green boxes. Absolutely conserved
residues in all sequences are marked in black. Residues in direct contact with Pm inhibitors are indicated
below the alignment as follows: azoxystrobin (red dots), myxothiazol (red triangles), MOAS (red squares).
Residues in direct contact with Pf QoIs are indicated below the alignment as follows: famoxadone (blue
squares), stigmatellin (blue dots), UHDBT (blue triangles), NQNO (blue stars), atovaquone (blue crosses).
Importantly, the 2 binding niches of cytb Qo differ from each other in QoI binding amino acid
residues (Figure 1.7) and in orientation upon QoI binding due to the conformational change of cytb
realised by the cd1 helix and PEWY motif. These two structural motifs of the cytb undergo large
movements upon binding of QoI (Figure 1.6), and are highly conserved among metazoans, plants,
fungi, protozoans and bacteria. Especially the PEWY motif is completely unchanged in all
eukaryotic and prokaryotic cytb (and cytb6) sequences examined so far (Figure 1.7). Three highly
conserved residues from the cd1 helix (G142, V145 and I146 bovine numbering) appear critical for
the class of Pf inhibitors, whereas two PEWY residues (P270 and E271), are more important for the
class of Pm inhibitors (Esser et al. 2004). Most interestingly, the residues I146 (I147 in yeast, I136
in mite) and P270 (P271 in yeast, P262 in mite) of the cd1 helix and PEWY motif respectively are
of special importance. They are both critical for the conformational change of cytb upon inhibitor
binding as they are in contact with all tested inhibitors so far: the Pm inhibitors azoxystrobin,
myxothiazol, MOAS, and the Pf inhibitors famoxadone, stigmatellin, UHDBT, NQNO and
atovaquone (Figure 1.7). Inhibitor binding generally causes P270 of the conserved PEWY motif to
back away in a unidirectional fashion from the Qo pocket, whereas the direction in which the cd1
helix moves clearly depends on the type of bound inhibitors: Pm inhibitors cause a negative shift
(relative to the inhibitor-free enzyme, defined as “off” position), whereas Pf inhibitors cause a
positive displacement (defined as “on” position). Although only a limited number of residues on the
cd1 helix are in contact with QoIs (Figure 1.7), the entire helix undergoes this inhibitor-type
dependent translation (Xia et al. 2007). Moreover, I146 seems to act as a substrate sensor and it
transmits an inhibitor-type dependent signal along the cd1 helix that serves as a switch for the
release or capture of the ISP-ED (Figure 1.6). Due to this intrinsic translation of the cd1 helix and
the PEWY motif upon inhibitor/QH2 binding, the ISP undergoes a conformational switch assumed
to be an intrinsic property of the bc1 complex as well (Esser et al. 2006; Figure 1.6). Interestingly, in
response to the displacements of the whole cd1 helix upon inhibitor/QH2 binding, the highly
conserved residue S151 (S152 in yeast, S141 in mite) undergoes significant positional adjustments
causing a single H-bond to the conserved residue K287 (K288 in yeast, K279 in mite).
Consequently, K287 is in turn stabilized to form 2 H-bonds to the backbone atoms of the ISP-ED
fixing it. Although not confirmed in bacterial bc1 mutants, S151 seems to interact with K287 to

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provide a more stable ISP-ED docking site in order to turnover properly electrons from QH2 to ISP
and cytc1 (Esser et al. 2006).
2.3.2 QoI insecto-acaricides (Insecto-acaricidal inhibitors of mitochondrially
encoded cytb Qo)
Acequinocyl, BTG505, atovaquone, fluacrypyrim, HNPC-3066 and hydramethylnon differ from
METI acaricides and other acaricides targeting mitochondrial respiration by the place of action in
the mitochondrial electron transport chain, as these acaricides are inhibitors of cytb Qo of complex
III (Dekeyser 2005; Krämer and Schirmer 2007), thus binding to different places in the structure of
the complex. Acequinocyl (3-dodecyl-1,4-dihydro-1,4-dioxo-2-naphtylacetate) is a pro-acaricide
which has to be metabolized into the active compound hydroxyacequinocyl (Figure 1.8) before
targeting cytb Qo. It is a hydroxy-naphthoquinone (HONQ), which primarily operates as a contact
acaricide, and is toxic for all developmental stages of spider mites (Kinoshita et al. 1999). Other
HONQ compounds are BTG505, the de-O-acetylated active compound of the pro-insecticide
BT504 (2-O-acetyl-3-(2-methylbut-3-en-2-yl) naphthalene-1,4-dione), and atovaquone (2-[trans-4-
(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-hydroxynaphthoquinone) (Figure 1.8). This latter
compound was primarily used as antiprotozoal control agent against the malaria vector Plasmodium
falciparum (Kessl et al. 2003, 2004), but shows also action against the pneumonia pathogen fungus
Pneumocystis jiroveci (Kessl et al. 2003), and other apicomplexan parasites Toxoplasma spp.,
Theileria spp. and Babesia spp. (Böhm et al. 1981; Araujo et al. 1991; Hughes and Oz 1995). Most
interestingly, it proves to be highly toxic to insects and spider mites (Schuster et al. 1979; Jacobsen
et al. 1986).
Fluacrypyrim (methyl(E)-2-{α-[2-isopropoxy-6-(trifluoromethyl) pyrimidin-4-yloxy]-o-tolyl}-3-
methoxyacrylate), the first strobilurin analogue of the MOA group used as acaricide, was originally
commercialised as a fungicide. It acts on all stages of tetranychids (Dekeyser 2005). Recently,
another strobilurin analogue of the MOA group, HNPC-A3066 ((E)- methyl 2-{2-[[[(Z)[1-(3,5-
bis(trifluoromethyl)phenyl)-1-methylthiomethylidene]amino]oxy]methyl]phenyl}-3
methoxyacrylate; Figure 1.8), was synthesized starting from 2 leading strobilurin compounds
fluacrypyrim and metominostrobin. This compound mainly showed larvicidal activity to T. urticae,
P. citri and P. ulmi, but was, to a slightly lesser extent, also effective against eggs and adult mites
(Liu et al. 2009; Pei et al. 2009). More new compounds of this group with acaricidal properties are
expected in the near future (Li et al. 2010). Another complex III inhibitor is hydramethylnon (5,5-

Chapter 1 ···············································································································
-33-
dimethyl-perhydro-pyrimidin-2-on-α-(4-trifluormethylstyryl)-α-(4-
trifluormethyl)cinnamylideenhydrazon) (Figure 1.8), also known as AC 217,300, belonging to the
trifluoromethyl aminohydrazones group. It is used primarily as an insecticide in the form of baits
for cockroaches and ants, but it has not been used in crops as it is not yet allowed in Europe (Table
1.2).
Figure 1.8 Chemical structure of insecto-acaricides inhibiting mitochondrial complex III (Caboni et al.
2004; Liu et al. 2009; Kurosu et al. 2010; Woodland 2010)
2.4 Bifenazate
Bifenazate is an ortho-biphenylcarbazate, with a methoxybiphenyl substituent on the terminal
nitrogen atom of the isopropyl carbazate (Figure 1.9). It is the only acaricide in the carbazate group,
and proves to act very selective on certain genera of spider mites (Tetranychus spp., Panonychus
spp., Oligonychus spp., Eotetranychus spp.). It is much less toxic to eriophyoid, tarsonemid and
tenuipalpid mites, and almost not toxic to pollinator insects and mammals (Kim and Seo 2001; Kim
and Yoo 2002; James 2002; Grosscurt and Avella 2005; Kim et al. 2005; Mommaerts et al. 2010).

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However, it was reported that adult females of T. occidentalis, treated with bifenazate, could not
produce any progeny after bifenazate treatment (Irigaray and Zalom 2006). In spider mites, it is
highly toxic to juvenile stages and adults, but slightly less effective on eggs. Bifenazate treatment is
characterised by fast knockdown effect and long residual action at temperatures in the range
between 15-35 °C (Pree et al. 2005; Ochiai et al. 2007). Initially, due to its paralysing effect, it was
assumed to act as a neurotoxin on the GABA-gated membrane chloride channel (Dekeyser 2005;
Krämer and Schirmer 2007). However, another mode of action as QoI and limited cross-resistance
to other QoIs will be shown in this thesis. Bifenazate is a pro-acaricide, which has to be activated by
hydrolysing its ester links by carboxylesterases (COEs). Consequently, S,S,S-
tributylphosphorotrithioate (DEF), organophosphates (OPs) and carbamates (CARBs) as inhibitors
of hydrolytic esterase activity, may act antagonistically to toxicity of bifenazate (Van Leeuwen et
al. 2006, 2007). Bifenazate, acequinocyl and fluacrypyrim are discussed more in detail in the
following chapters.
Figure 1.9 Chemical structure of bifenazate (Woodland 2010)
3. Spider mite resistance to insecto-acaricides
“Whereas the presence of resistance was a rare phenomenon during the early 1950s, it is the fully
susceptible population that is rare in the 1980s. Unquestionably the phenomenon of resistance
poses a serious obstacle to efforts to increase agricultural production and to reduce or eliminate
the threat of vector-borne diseases” (Georghiou 1986)

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