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The role of horizontally transferred genes in the xenobiotic adaptations of the spider mite Tetranychus urticae

Wybouw, N.R.

Publication date 2015 Document Version Final published version

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Citation for published version (APA): Wybouw, N. R. (2015). The role of horizontally transferred genes in the xenobiotic adaptations of the spider mite Tetranychus urticae.

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General Introduction

1.1. ARTHROPOD HERBIVORY ased on fossil records dating back to the Late Silurian era (420 million years Bago), arthropods were the first terrestrial animals that showed signs of herbivory (or feeding exclusively on plant material) (Labandeira, 2002, 2006). Arthropods are characterized by the presence of articulated appendages (arthropod means ‘jointed foot’) and a chitinous exoskeleton, which necessi- tates ecdysis for growth. This phylum contains four living subphyla: Myriapoda, Crustacea, Hexapoda and Chelicerata (FIGURE 1.1) and represents a great majority of all currently living plant-feeding animal species (Schoonhoven et al., 2006; Strong et al., 1984). Although every subphylum harbors phytophagous species, only the Hexapoda and Chelicerata lineages will be discussed. Arthropod herbivory within these subphyla can be divided into four different feeding guilds: (1) external feeding (chewing, snipping or tearing), (2) piercing-sucking (cell-content, xylem or phloem), (3) internal feeding by creating tunnels between upper and lower plant surfaces in a process called mining, and (4) feeding through gall formation or other plant distortions. Typically, a phytophagous arthropod lineage is characterized by one particular mode of feeding. For instance, within insects, beetles and caterpillars are chewing herbivores, while some true bugs (or Hemiptera) pierce and suck plant tissue. Within Chelicerata, plant-feeding mites typically pierce and suck cell content (Labandeira, 2005; Lindquist, 1998; Schoonhoven et al., 2006).

1.1.1. THE CHALLENGES OF ARTHROPOD HERBIVORY Remarkably, even though plants represent the most readily available and abun- dant food source in terrestrial ecosystems, only a limited number of chelicerate and hexapod lineages adopted a phytophagous lifestyle. Within Hexapoda, herbivory is only detected in nine living orders (Labandeira, 2002; Schoonhoven et al., 2006; Strong et al., 1984), while in Chelicerata, and more specifically, in the terrestrial class of the Arachnida, adaptations to obligate phytophagy mainly occurred in the order of the Trombidiformes which belongs to the subclass of mites and ticks (or the Acari) (Krantz & Lindquist, 1979; Lindquist, 1998) (FIGURE 1.1). It is argued that only a restricted number of arthropod lineages were able to develop a phytophagous lifestyle because the unique composition of land plants makes them arduous and unfavorable food. First, plant cells are enclosed in a recalcitrant cell wall which arthropods can find hard to penetrate due to its

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Chapter 1 ss- Dated evolutionary history of are based on a combination of the Arthropoda Time line and phylogeny phylum. sources 1.1. IGURE (Dabert et al., 2010; Hedges & Kumar, 2009; Lindquist, 1998; Regier et al., 2010; Yeates et al., 2012). Divergence times within et al., 2012). Divergence et al., 2010; Yeates 2009; Lindquist, 1998; Regier (Dabert et al., 2010; Hedges & Kumar, Clades posse in blue. are marked LineagesAcariformes within the Hexapoda and Chelicerata subphyla debate. are still under much their names printed in bold green font. ing phytophagous species have F

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mechanical strength as well as to digest due to an inherently inadequate battery of enzymes to break down cellulose, lignin or pectin (Carpita & Gibeaut, 1993; Heredia, 2003; Sorensen et al., 2010; Whetten & Sederoff, 1995). Second, arthropods and plants have a distinctly different chemical composition, characterized by a relative high protein and carbohydrate content, respectively (Mattson, 1980; Strong et al., 1984). Another evolutionary hurdle for plant feeding arthropods is thus to elevate between what they nutritionally need for their own development and what plant tissues offer in terms of nutrients. Third, once arthropods (and other organisms) evolved to access and feed on plant tissue, host plants counteracted by developing anti-herbivore defenses. Plant resistance to herbivores is achieved by physical barriers (which include tri- chomes, spines and rough leaves) and/or by producing chemical defenses (Howe & Jander, 2008). These defensive phytochemicals (or ‘plant allelochemicals’) can both repulse and poison herbivores or interfere with the assimilation of plant nutrients inside the herbivore’s gut (Whittaker & Feeny, 1971). These are only a few of many evolutionary obstacles arthropods face on their road to herbivory. However, once an arthropod species successively underwent the necessary adaptive breakthroughs (or key innovations), it is thought that the acquired ability to colonize a new plant or plant family can promote massive species diversifications (Mitter et al., 1988). Indeed, within the Hexapoda subphylum, the herbivorous lineages belong to the most species rich orders, with the Coleoptera and Lepidoptera (349,000 and 119,000 species, respectively) on top (Labandeira, 2002; Schoonhoven et al., 2006; Strong et al., 1984). In parallel, two obligate phytophagous Acari superfamilies (Tetrany - choidea and Eriophyoidea) are also exceptionally species rich (Krantz & Lindquist, 1979; Lindquist, 1998).

1.1.2. HOST PLANT RANGE OF ARTHROPOD HERBIVORES Depending on the potential host plant species range, arthropod herbivores are traditionally divided into specialists (which exhibit mono- and oligophagy) and generalists (which are polyphagous). FIGURE 1.2 depicts some example species along the scale from being strictly monophagous to polyphagous. In contrast to specialists, generalist herbivores are able to feed and develop on plants belonging to unrelated, phylogenetically distinct plant families. Within arthropods, there has been an evolutionary trend to specialize to specific host plants (Nosil, 2002; Schoonhoven et al., 2006), making true polyphagous herbivores scarce. Indeed, less than 10% of all phytophagous insects are able to feed on plants of

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more than three plant families (Bernays & Graham, 1988). However, these rare polyphagous herbivores can be found in diverse orders, both within Hexapoda and Chelicerata (Krantz & Lindquist, 1979; Lindquist, 1998; Schoonhoven et al., 2006; Strong et al., 1984).

1.2. PHYTOPHAGOUS MITES Mites are placed in the Acari subclass within the largest chelicerate class of the Arachnida. They can be distinguished from their closest chelicerate and arachnid relatives by their unique body plan, small size (never exceeding a few centimeters) and great diversity in life styles. The mite body consists of one single segment and is artificially divided into the gnathosoma, the feeding region holding the primary organs of food acquisition (e.g. chelicerae and pedipalps), and the idiosoma, which is the site for all other life functions, e.g. locomotion, post-oral digestion, and reproduction. Mites adapted to a diverse range of ecological niches and diversified the structure of their chelicerae allowing them to feed on plants, bacteria, animals and fungi (Walter & Proctor, 1999). Although a handful of mite species in the Sarcoptiformes order can be classified as herbivores, adaptations to obligate phytophagy predominantly occurred in the order of the Trombidiformes and have led to three major phytophagous mite clades: Eriophyoidea, Tetrany- choidea, and Tarsonemidae of which the latter belongs to the Heterostigmata lineage (Krantz & Lindquist, 1979; Lindquist, 1998) (FIGURE 1.1).

1.2.1. BIOLOGY AND MORPHOLOGY OF PHYTOPHAGOUS MITES The chelicerae of phytophagous mites are modified into a stylet-like structure allowing the mites to feed from plants by piercing plant cells and sucking up cell- content. Tarsonemid, tetranychoid and eriophyoid mites are morphologically distinguishable from one another. While tetranychoid mites exceed 400 µm, the size of eriophyoid and tarsonemid mites only ranges between 90 and 350 µm. Adult eriophyoid mites have a more elongated worm-like body with only two pairs of legs, while adult tetranychoid and tarsonemid mites have four pairs. In tarsonemid males, the fourth pair of legs is extensively modified and used to carry a quiescent female, prior to mating (Walter & Proctor, 1999; Zhang, 2003). An additional characterizing mite trait is body colour. While eriophyoid and tarsonemid mites are pale yellow to white, the former does not accumulate plant chlorophyll inside their bodies. In tetranychoid mite species, green and red color morphs exist (Helle & Sabelis, 1985; Ueckermann, 2010; Zhang, 2003).

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Phytophagous mites typically have exponential population growths and inflict significant damage to their host. These mites easily disperse to new host plants by passively drifting on wind currents. Additionally, some tarsonemid and eriophyoid mites are also known to attach to other animals for transport (also called zoonotic transport) (Helle & Sabelis, 1985; Ueckermann, 2010; Zhang, 2003). Despite that arthropod predators are deployed in greenhouses to combat mite infestations, the control of phytophagous mites is still mainly based on acaricides (Knowles, 1997; Van Leeuwen et al., 2014).

1.2.2. SPIDER MITES Within Tetranychoidea, the approximately 1200 species of the Tetranychidae family are commonly called spider mites. The name ‘spider mite’ refers to their ability to produce silk-like webbing which serves many biological functions, including predator defense and intraspecific communication (Clotuche et al., 2012; Clotuche et al., 2013; Helle & Sabelis, 1985). Spider mites are haplo- diploid and have an arrhenotokous parthenogenic mode of reproduction, meaning that unfertilized diploid females only produce haploid male offspring. After egg hatching, the life cycle of spider mites consists of four active feeding stages: larva, protonymph, deutonymph and adult. The last three feeding stages include intermittent quiescent phases: protochrysalis, deutochrysalis and teliochrysalis (Helle & Sabelis, 1985). Spider mites can feed through the spongy plant parenchyma (adult Tetranychus urticae mites have a stylet of about 160 µm long) which typically results in foliar chlorotic spots and eventually leads to leaf necrosis and abscis- sion (Helle & Sabelis, 1985; Jeppson et al., 1975; Park & Lee, 2002). Spider mite species cover the complete spectrum from specialist to generalist. For instance, Tetranychus lintearius (or the gorse spider mite) and Mononychellus progresivus feed exclusively on gorse (Ulex europaeus) and cassava (Manihot esculenta), respectively (Hill & Odonnell, 1991; James, 1988; Navajas, 1998). Tetranychus evansi (or the red tomato spider mite) is able to infest different plant species as long as they belong to the Solanaceae plant family (Demoraes et al., 1987). At the opposite end of the spectrum is T. urticae (the two-spotted spider mite), the study species of this work, which successfully feeds on over 1000 plants which are scattered over more than 140 distinct plant families (Jeppson et al., 1975; Migeon & Dorkeld, 2015) (FIGURE 1.2). Tetranychus urticae is one of the most polyphagous herbivores and causes major agricultural and horticultural losses worldwide (Jeppson et al., 1975; Van Leeuwen et al., 2010). Experimental

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FIGURE 1.2. Arthropod herbivores scaled based on their potential host plant range, going from monophagy (specialists) to polyphagy (generalists). The number of docu- mented host plants per arthropod herbivore is shown between brackets after the species’ name. The insect photos were redrafted from Barrett and Heil (2012) and the T. urticae photo was taken by J. van Arkel.

evolutionary studies show that T. urticae populations rapidly adapt to new host plants (Agrawal, 2000; Fry, 1989; Gould, 1979) and infer that this high adaptive potential is (partially) due to its great intraspecific genetic variability (Magalhaes et al., 2007). Tetranychus urticae has been dubbed ‘the most resistant species’ owing to the record number of pesticides to which populations developed resistance (Van Leeuwen et al., 2010) (http://www.pesticideresistance.org/).

1.3. ARTHROPOD GENOMICS AND TRANSCRIPTOMICS The landmark technological discovery of Sanger et al. (1977) and more recently developed technologies (referred to as ‘Next-Generation Sequencing’ (NGS) technologies) allow to sequence and analyze genomes (or complete DNA sets) of organisms. Due to their easy laboratory rearing, short generation times and often small genome sizes (the 90 Mb T. urticae genome was predicted to be the smallest arthropod genome; Hanrahan & Johnston, 2011), genomes of arthropods have been favoured for sequencing experiments (Evans et al., 2013). Indeed, a survey done by Song & Wang in 2013 shows that over 30% of the completely sequenced non-human animal genomes belongs to arthropod species. Moreover, projects such as the ‘i5k’-initiative keep producing fully sequenced arthropod genomes at a high rate (Evans et al., 2013; Robinson et al., 2011). This genomic sequence data has opened up new inroads for studying arthropod herbivory. For instance, by comparing the genomes of closely related herbivorous and non-herbivorous lineages, genes can be identified which

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might have contributed to a successful transition towards herbivory in those groups (Gloss et al., 2014; Goldman-Huertas et al., 2015). In addition, these modern technologies also allow for profiling the genome- wide gene-expression levels (also called transcriptome) in a specific organism, organ or tissue and can unbiasedly detect differential expression which corre- lates with abiotic or biotic parameters. Multiple transcriptomic technologies exist (one- and two-color gene-expression (GE) microarrays, RNA-seq) of which the experimental designs depend on whether the organism has a fully sequenced genome or not. In RNA-seq protocols, with an available genome as a reference, sequenced transcripts can be ‘mapped’ and counted to previously annotated gene sequences. Without an available genome sequence, a reference transcriptome should first be assembled de novo using the library of all sequenced transcripts (Boerjan et al., 2012; Oppenheim et al., 2015). In GE microarrays, oligonucleotide probes are synthesized that are complementary to available sequence data and are spotted on a glass array at specific locations. RNA samples are then fluorescently labeled and hybridized to these complementary probes. The fluorescent intensities which arise from the transcript- probe hybridizations mirror gene-expression levels and are used for the relative quantification of gene expression ratios (Malone & Oliver, 2011). Transcrip - tome sequencing can be favoured over genome sequencing due to its lower sequencing costs and more straight forward sequence analysis (Riesgo et al., 2012). Genome sequencing on the other hand allows for a better distinction between potential allelic variants and paralogues. In parallel with the aim of the ‘i5k’-project to sequence 5000 arthropod genomes, the ‘1kite’-project plans to produce assembled transcriptomes of over 1000 insect species (www.1kite.org). Similarly to genome sequencing projects, transcriptomic studies also signif- icantly contribute to our understanding of arthropod herbivory. For example, by selectively sequencing gene transcripts expressed in the midgut of plant feeding beetles, new digestive enzymes were identified that are able to metabolize complex plant compounds (Kirsch et al., 2014; Pauchet et al., 2009, 2010). Surprisingly, a subset of these enzymes are not present in other arthropod lineages and have closest homology to microbial enzymes which led some to con- clude that these are in fact coded by sequenced genes from symbiotic bacteria residing in the arthropod digestive tract. Symbionts are crucial for the digestion of recalcitrant plant food in some arthropod species (Watanabe & Tokuda, 2010).

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1.4. ENZYMATIC COUNTERMEASURES OF ARTHROPODS TO CHEMICAL PLANT DEFENSES

Arthropod herbivores have to deal with the plant produced xenobiotics and have in turn developed a multitude of ways to safely handle plant defenses. These adaptive responses are considered major evolutionary drivers in plant- arthropod interactions and are traditionally divided into three categories: behavioral, physiological and biochemical (or enzymatic) (FIGURE 1.3) (Brattsten, 1988). Behavioral counter-adaptations generally lead to feeding patterns that avoid allelochemical exposure. For instance, some insects developed a vein-cutting behavior which drains defensive phytochemicals from their feeding site (Dussourd & Eisner, 1987). Typical physiological adaptations preclude the toxic allelochemical from reaching its target tissue and include pH adjustments and formation of specialized body compartments (Hartmann, 1999; Pentzold et al., 2014) (FIGURE 1.3A). Although traditionally categorized as physiological adaptations, these counter-defenses often reflect a set of numerous distinct biochemical and enzymatic adaptations. In the next sections and throughout my thesis, I will focus on the enzymatic adaptations of arthropods to plant allelochemicals. However, this does not imply that this is the most important line of arthropod defense against toxic phytochemicals.

1.4.1. THE XENOBIOTIC METABOLISM OF ARTHROPOD HERBIVORES 1.4.1.1. Ubiquitous enzymes that bind, metabolize and transport toxins Once a plant toxin has entered the herbivore’s system, the herbivore can inter- act with it using enzymes of its xenobiotic metabolism. Xenobiotic metabolism can be divided into 3 phases: metabolization (phase I), conjugation (phase II) and translocation (phase III) (Brattsten, 1988) (FIGURE 1.3B). Enzymes which catalyze these reactions typically belong to ubiquitous multi-gene families. Cytochrome P450 monooxygenases (or CYPs) are Phase I enzymes and play an essential role in the adaptation of herbivores to plant defenses (Feyereisen, 1999). CYP enzymes contain a heme cofactor and catalyze the transfer of one O-atom of molecular oxygen to the substrate, while reducing the other atom to water. CYP enzymes attack a wide range of xenobiotics and, depending on the substrate structure, will act as isomerases, reductases or oxi- dases (Ahmad, 1986; Feyereisen, 1999, 2011). Another example of Phase I enzymes are carboxyl/choline esterases (or CCEs) which detoxify toxins by hydrolyzing covalent bonds. Both in insect and mite herbivores, CCE and CYP

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FIGURE 1.3. A: The different multi-layered herbivore strategies to overcome chemical plant defenses. (1) The first step is avoiding internalization of toxic allelochemicals which can be achieved by behavioral adaptations (e.g. vein-cutting (Dussourd & Eisner, 1987)), physiological modifications (e.g. pH adjustments in the digestive tract which limit bioactivation of allelochemicals (Pentzold et al., 2014)) and enzymatic adaptations (e.g. deactivating the allelochemical in plantae (Musser et al., 2002)). (2) After internalization, the allelochemical can be enzymatically detoxified and actively (enzymatic) or passively (physiological) excreted. (3) By a distinct set of adaptations (traditionally referred to as physiological processes), high concentrations of allelochemicals can be safely stored in specialized internal compartments in a process called sequestration. (4) Mutations in the target of the allelochemical can minimize its deleterious effects. B: Enzymatic adaptations within the xenobiotic metabolism of herbivores. Xenobiotic metabolism handles an internalized allelochemical in 3 separate phases: metabolization, conjugation and translocation. Enzymes of different phases often work in unison in order to completely eliminate the poisonous effects of an allelochemical. Through the enzymatic actions within phase I and II, lipophilic xenobiotics become more water sol- uble and easier excreted. Target site mutations are not included in these enzymatic adaptations.

activity highly varies when fed on different host plants suggesting that particular components of a plant’s phytochemical profile act as a blend of inducers (Ahmad, 1986; Feyereisen, 1999). Phase II enzymes catalyze the conjugation of the products of the phase I reactions with another molecule, making them more hydrophilic. However, in many cases these phase II enzymes will also directly conjugate the ingested xenobiotic to another molecule. UDP-glucosyltransferases (or UGTs) catalyze the conjugation of lipophilic molecules with uridine diphosphate (or UDP)-glucosides. Arthropod UGTs have long been implicated for playing a major role in the detoxification process of plant allelochemicals. For example, benzoxazinoids are defense compounds of Poaceae (grasses) which, mediated by β-glucosidases, produce toxic aglucones which negatively affect the reproductive per- formance of many lepidopterans. However, the moth Spodoptera frugiperda (or the fall armyworm) adapted to this plant defense mechanism by re-glucosylat- ing the poisonous products into recalcitrant benzoxazinoids that are unaffected by both plant and insect β-glucosidases (Wouters et al., 2014). Glutathione S- transferases (or GSTs) are another group of phase II enzymes which conjugate reduced glutathione with (metabolized) toxicants. GSTs often work in concert with CYP enzymes by conjugating their epoxide reaction products. The produced glutathione S-conjugates are more hydrophilic and thus are easier excreted from the herbivore’s body (Brattsten, 1988; Lee, 1991).

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Although originally largely neglected, phase III enzymes, which translocate chemicals across membranes, are now considered as key components in a herbivore’s xenobiotic metabolism (Sorensen & Dearing, 2006). Lepidopteran studies have shown that multidrug ATP Binding Cassette (or ABC) transporters prevent phytotoxins from getting into contact with their target tissue and translocate them to organs that eliminate toxins from the insect body (Gaertner et al., 1998; Petschenka et al., 2013). Due to the multiplicity of enzymes within each family, the individual cat- alytic abilities of each paralogue are difficult to identify using classical biochemical and molecular techniques. However, functional genomics tools now provide a new inroad by characterizing the exact sequences of all genes and the enzymes they encode.

1.4.1.2. The evolutionary history of the ubiquitous enzymes of the xenobiotic metabolism Genetic studies in highly adapted Papilio butterflies uncovered duplicated CYP genes (Wen et al., 2006). Comparative genomics of the xenobiotic metabolism is now uncovering that these duplication events are a common trend for insect herbivores and often lineage-specific. For instance, compared to their respective non-phytophagous relatives, the phytophagous beetle Dendroctonus ponderosae and dipteran Scaptomyza nigrita proliferated their CYP and GST gene repertoire, respectively (Gloss et al., 2014; Keeling et al., 2013). Gene duplications can lead to subfunctionalization and neofunctionalization events (Conant & Wolfe, 2008). Subfunctionalization occurs when the duplicated daughter paralogues specialize in only a fraction of the ancestral biological function, while neofunctionalization involves duplicated paralogues able to catalyze entirely novel reactions. In insects, functional analysis of duplicated paralogues now shows that these two processes also occur within the xenobiotic metabolism and facilitate adaptation to chemically challenging hosts (Gloss et al., 2014; Wen et al., 2006). In addition to identifying the complete set of paralogues, genomics can also give clues to how a phytophagous arthropod diversified its gene repertoire. In the genome of the moth Plutella xylostella (or diamondback moth), a higher density of transposable elements (or TEs) were located near diversified CYP paralogues involved in detoxification pathways compared to CYP genes serving a function in insect development. TEs are repetitive DNA elements which promote unequal crossing over and could thus have caused the duplication of the neigh- boring CYP genes (You et al., 2013).

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Exposure to allelochemicals often induces the tissue-specific production of counteracting detoxifying enzyme(s) in adapted herbivores, allowing transcriptomics to further direct us towards the exact specialized paralogue which is causing herbivore immunity. For instance, using GE microarray data, Bass et al. (2013) showed that the aphid Myzus persicae (or green peach aphid) adapts to tobacco, which produces the toxic alkaloid nicotine, by amplifying and over- expressing CYP6CY3. Guided by a gland-specific cDNA library in the poplar leaf beetle, Chrysomela populi, a particular ABC transporter was identified which proved essential for the sequestration process of the phytochemical salicin from its haemolymph into the defensive glands. By sequestering salicin into its exocrine glands, C. populi produces defensive secretions to deter predators (Strauss et al., 2013). Unfortunately, even though early studies indicated that insects and mites evolve resistance to plant toxins using different strategies (Mullin & Croft, 1983), the great majority of genomic and transcriptomic research on arthropod xenobiotic adaptation was focused on insects.

1.4.1.3. Recruitment of novel enzymes in the insect xenobiotic metabolism Through neo-functionalization, gene duplication can supply herbivores with new, non-ubiquitous enzymes to dismantle plant defense pathways. Insect studies have shown that lineages can independently evolve the same enzyme by duplicating similar genes. For instance, both moths and grasshoppers independently recruit- ed a pyrrolizidine-alkaloid-N-oxygenizing enzyme (PNO) in their xenobiotic metabolism by duplicating a flavin monooxygenase. PNO detoxifies pyrrolizidine alkaloid allelochemicals by oxidizing its toxic free base into its non-toxic N-oxide (Wang et al., 2012). Alternatively, through neo-functionalization, insects also evolved different novel enzymatic strategies to detoxify the same plant defense mechanism. Upon herbivore attack, the Brassicales plant defense system releases toxic isothiocyanates from glucosinolates by mixing these thioglucoside precur- sors with hydrolyzing myrosinase enzymes. P. xylostella and Pieris butterflies adapted to this glucosinolate-myrosinase defense system by evolving a unique glucosinolate sulfatase enzyme that biotransforms the glucosinolates precluding myrosinase degradation and a unique nitrile-specifier protein that redirects the glucosinolate hydrolysis to less toxic nitrile products, respectively (Fischer et al., 2008; Ratzka et al., 2002; Wheat et al., 2007; Wittstock et al., 2004).

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1.4.2. HERBIVORES MANIPULATE INDUCIBLE PLANT DEFENSES Plant allelochemicals can be divided into two groups based on their production. Phytoanticipins are allelochemicals that are produced at a constant rate and are therefore always present in a plant tissue. In contrast, phytoalexins are compounds of which the production is induced upon receiving a certain herbivore attack stimulus (Vanetten et al., 1994). Phytoalexin production is mainly gov- erned by three phytohormones: jasmonic acid (or JA), salicylic acid (or SA) and ethylene (or ET). These signaling pathways are differentially induced, depending on the functional feeding guild of the attacking herbivore. Chewing herbivores typically induce JA-dependent pathways (Howe & Jander, 2008), while mainly SA-dependent pathways are up-regulated upon phloem-feeding activity (Thompson & Goggin, 2006). Cell-content sucking mites induce both JA- and SA-pathways (Alba et al., 2015; Kant et al., 2008; Zhurov et al., 2014). Studies indicate that in addition to developing tolerance or resistance to phytoalexins, a wide range of arthropod herbivores also adapt by manipulating the inducible production of these compounds (Musser et al., 2002; Sarmento et al., 2011; Zarate et al., 2007). By secreting small molecules (in case of proteins, called ‘effectors’) via their saliva into their host plant, arthropod herbivores interfere with the plant signaling cascade, leading to an attenuation or even suppression of phytoalexin production (Alba et al., 2011). The first identified arthropod effector was the Helicoverpa zea glucose oxidase enzyme that suppresses the production of the poisonous allelochemical nicotine (Musser et al., 2002). Unfortunately, in contrast to the well characterized effectors of microbial plant pathogens, the exact identities and functions of arthropod effectors remain elusive. However, as the intangible arthropod effectors are secreted into the plant via the saliva, salivary gland transcript libraries are predicted to harbor their nucleotide sequences. Indeed, a screening pipeline, developed by Bos et al. (2010), mined the proteomes of multiple M. persicae gland libraries for specific attributes and identified 46 candidates, with some proteins showing indications of effector activity.

1.4.3. ENZYMATIC ADAPTATIONS TO POLYPHAGY IN ARTHROPOD HERBIVORES As different plant families have distinct allelochemical profiles (Harborne & Baxter, 1999; Hartmann, 1999), it remains puzzling how those scarce polypha - gous arthropod species cope with the myriad of different sets of phytotoxins.

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Krieger et al. (1971) originally conjectured that generalists developed a greater enzymatic capacity than specialists to detoxify these varying plant toxins. However, the great inducibility of detoxifying enzyme activity in herbivorous arthropods upon different hosts renders the Krieger hypothesis inherently impossible to prove false (or ‘unfalsifiable’) (Ahmad, 1986; Brattsten, 1988). Since then, the theory was reformulated stating that generalists could have evolved a xenobiotic metabolism with a broader substrate range. As specialists have a more predictable blend of dietary allelochemicals, they are expected to develop a more efficient detoxification mechanism of specific toxins, compared to generalists. Indeed, although CYPs of a lepidopteran generalist metabolize a wider range of plant toxins than those of a lepidopteran specialist, the generalist’s enzymes exhibit lower activity levels (Li et al., 2004). However, this ‘jack-of-all-trades, master of none’ theory is not fully resolved yet as some studies seem to disprove it (Ali & Agrawal, 2012) and, especially at the molecular level, more studies are needed to investigate how exactly generalists cope with the abundance of plant toxins.

1.4.4. ENZYMATIC DETOXIFICATION OF PESTICIDES IN ARTHROPOD HERBIVORES In his seminal paper of 1961, Gordon argued that, in the eyes of an arthropod herbivore, a man-made chemical pesticide can be seen as a newly evolved plant chemical and that herbivores co-opt their pre-evolved phytochemical detoxifying systems to detoxify these synthetic toxins. Some studies indeed support a link between host plant adaptation and pesticide resistance (Ahmad, 1986; Berry et al., 1980; Lindroth, 1989; Sen Zeng et al., 2007) and, using a genomics approach, successfully identified specific paralogues of multi-gene families traditionally associated with xenobiotic metabolism that are capable of detoxifying pesticides (Bass et al., 2013; Joussen et al., 2012). Within this theoretical framework, this means that the evolutionary response of polyphagous herbivores to the diverse range of dietary plant toxins preadapts them to the exposure of various pesticides. From Gordon (1961), I paraphrase: ‘the extraordi- nary high and generalized tolerance of the feeding stages of polyphagous arthropods to contact pesticides is probably the result of selection for endurance of prolonged and varied biochemical stresses associated with the diversity of their natural food plants’. Although an appealing theory, some nuances need to be considered as the selection pressures, arising from the two toxin classes, differ in both intensity and duration. While pesticides, when

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exposed to arthropod pests, are expected to have a uniform concentration of the same toxin, plant toxins are heterogeneously distributed in both space and time (Despres et al., 2007). Fine-tuning the ‘Gordon hypothesis’ thus calls for additional empirical data, especially at the molecular level (Rosenheim et al., 1996).

1.5. STUDY OF THE XENOBIOTIC METABOLISM IN PHYTOPHAGOUS MITES USING ACARI GENOMICS Regrettably, due to a lack of chelicerate model species (herbivorous as well as non-herbivorous), post-genomics studies were mainly guiding the dissection of plant-insect interactions and were neglecting plant-mite interactions. Addressing this issue, in 2007 the spider mite T. urticae was opted as a chelicerate model for its status as a polyphagous, worldwide pest and acaricide resistance champion as well as its easy laboratory rearing and predicted small genome size (Grbic et al., 2007). In 2011, the 89.6 Mb long genome of T. urticae was Sanger sequenced (assembled into 640 scaffolds) and was the first sequenced chelicerate genome (Grbic et al., 2011) (http://bioinformatics.psb.ugent.be/ orcae/overview/Tetur). Since then, the genomes of several non-herbivorous mites, ticks and other chelicerates have also been sequenced (TABLE 1.1) (Cao et al., 2013; Chan et al., 2015; Cornman et al., 2010; Evans et al., 2013; Guerrero et al., 2006; Hilbrant et al., 2012; Hill, 2010; Hoy, 2009; Robinson et al., 2011; Sanggaard et al., 2014). Compared to insect genomes, the ubiquitous detoxifying multi-gene families have a unique arrangement in T. urticae and show an unprecedented transcriptional induction upon phytochemical blends. The mite genome harbors 86 CYP genes of which 43 paralogues resulted from a T. urticae-specific expansion of the CYP2 clan. Genome annotation revealed that the T. urticae GST family is similarly expanded and includes a new arthropod group of Mu-class GSTs (Grbic et al., 2011). T. urticae also possesses a record number of multidrug ABC transporters of which the subfamilies ABCC, ABCG and ABCH are differentially expressed when mites are transferred from bean to Arabidopsis and toma- to (Dermauw et al., 2013). The annotated genomic sequence of this polyphagous mite pest now not only opens up new research avenues for unravelling plant-mite interactions but also for testing the preadaptation hypothesis of generalists towards pesticides on the molecular level (Gordon, 1961).

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TABLE 1.1. Sequenced chelicerate genomes, published before March 2015. Order Species Reference Araneae Latrodectus hesperus Robinson et al., 2011 Loxosceles reclusa Evans et al., 2013 Parasteatoda tepidariorum Hilbrant et al., 2012 Stegodyphus mimosarum Sanggaard et al., 2014 Acanthoscurria geniculate Sanggaard et al., 2014 Ixodida Ixodes scapularis Hill, 2010 Rhipicephalus microplus Guerrero et al., 2006 Scorpiones Centruroides sculpturatus Evans et al., 2013 Mesobuthus martensii Cao et al., 2013 Sarcoptiformes Dermatophagoides farinae Chan et al., 2015 Mesostigmata Metaseiulus occidentalis Hoy, 2009 Varroa destructor Cornman et al., 2010

1.6. HORIZONTAL GENE TRANSFER Horizontal gene transfer (HGT) is commonly described as the non-sexual transmission of genetic material across species boundaries (FIGURE 1.4A). As this research avenue will be a main focus of my thesis, I will discuss the process of HGT in more detail.

1.6.1. DETECTING HORIZONTAL GENE TRANSFER The golden standard of detecting HGT is showing that the phylogenetic tree based on the transferred gene is incompatible with the known species phylogeny. HGT can lead to different patterns of discrepancies depending on the ancestral distribution of the transferred gene. For instance, the horizontally transferred gene could be a completely novel gene without related homologues in the recipient or related species (FIGURE 1.4B, left). Alternatively, a gene copy could supplement or replace an existing homologue in the recipient genome through HGT (FIGURE 1.4B, middle and right, respectively). However, despite these different phylogenetic patterns, HGT is generally detected when the laterally acquired gene in the recipient species is embedded with strong node support in a group of homologues belonging to non-related donor species.

1.6.2. HORIZONTAL GENE TRANSFER IN MICROORGANISMS HGT is widespread and prevalent in prokaryotes where it operates through three distinct mechanisms: (1) transformation: direct uptake of exogenous DNA, (2) conjugation: plasmid-mediated uptake of foreign DNA through cell-

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FIGURE 1.4. A: The three known mechanisms of HGT in prokaryotes; transformation (direct uptake of foreign DNA), conjugation (plasmid-mediated uptake) and transduction (virus-mediated uptake). B: Three examples of how HGT can distort phylogenetic trees. Left: the horizontally transferred gene is a novel gene without related homologues in the recipient or related species. Middle: a gene copy supplements an existing homologue in the recipient genome. Right: a gene copy replaces an existing homologue in the recipient genome through HGT.

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to-cell contact, and (3) transduction: virus-mediated uptake of foreign DNA through phages (FIGURE 1.4A). By providing genomic innovation, HGT serves a double biological function in bacteria. First, by counteracting the accumulation of deleterious mutations over generations due to the microbial asexual reproduction, it serves a similar role as recombination in sexually reproducing eukaryotes (Felsenstein, 1974; Muller, 1964). Second, HGT promotes prokaryotic adaptation to new and changing environments. By means of HGT, microbes become tolerant to various antibiotics, shift to a pathogenic lifestyle and survive environmental extremes. Therefore, already since the 20th century, HGT is acknowledged as a major force in the adaptive evolution of prokaryotes (Koonin et al., 2001; Pal et al., 2005; Springael & Top, 2004).

1.6.3. HORIZONTAL GENE TRANSFER IN ANIMALS HGT studies were traditionally restricted to the prokaryotic domain of life as multicellular eukaryotic biology disfavors HGT. First, before genetic information can be integrated and expressed in a eukaryotic genome, it first has to pass through the selective double membrane of the nucleus. Second, if it concerns a bacterial donor, the mobile genetic element needs to be adjusted to the eukaryotic transcription machinery (polyadenylation, codon compositions and binding site modifications). Last, in multicellular organisms, a new genetic element can only be transmitted to the following generation if it is incorporated within the isolated germ cell line. Yet, surprisingly, despite these major evolutionary barriers, current studies indicate that HGT does continuously affect eukaryotic evolution (Crisp et al., 2015). Moreover, comparative genomics show that HGT even con- tributes to the genomic content of multicellular animals albeit with varying rates in different lineages (Andersson, 2005; Hotopp, 2011; Hotopp et al., 2007). The effect of HGT on animal evolution is a topic of heavy debate. Some argue that if genes are indeed horizontally transferred to animals, they are like- ly to deteriorate into pseudogenes and no longer code for functional proteins. Others argue that HGT to animals is a crucial driver in the evolutionary transition of its endosymbiotic bacteria to organelles (Hotopp, 2011). Some state that, as in prokaryotes, HGT might accelerate the adaptive evolution of animals and contribute selective advantages crucial for survival in novel and challenging environments (Hotopp, 2011; Hotopp et al., 2007; Kirsch et al., 2014; Moran & Jarvik, 2010).

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1.6.4. HORIZONTAL GENE TRANSFER IN T. URTICAE When analyzing the T. urticae genome, Grbic et al. (2011) uncovered a remark- able number of potentially horizontally transferred genes by showing that their best BLAST hits were proteins of microbial organisms (TABLE 1.2). Phylogenetic analysis showed that five of these T. urticae genes (tetur01g11260, tetur01g11270, tetur11g04810, tetur11g04820 and tetur11g04840) have homologues in certain hemipteran and dipteran lineages and that these are consistently acquired by horizontal gene transfer from a fungal donor species (Cobbs et al., 2013; Grbic et al., 2011). The enzyme products of these genes are identified as functional carotenoid cyclase/synthases and carotenoid desaturases and are indirectly connected to important arthropod traits such as defense, colour and vision (Grbic et al., 2011; Moran & Jarvik, 2010). Seventeen putatively horizontally transferred T. urticae genes code for intradiol ring-cleaving dioxygenases (TABLE 1.2). In bacteria and fungi, this family of dioxygenases catalyzes the oxygenolytic fission of monomeric and polymeric o-dihydroxyphenolic substances (Vaillancourt et al., 2006). As plants produce many toxic phenolic compounds in their defense against herbivores, Grbic et al. (2011) hypothesizes that the ability to metabolize these substances by the lateral acquisition of intradiol dioxygenases facilitates T. urticae to survive plant defenses. The precise microbial donor species and evolutionary history of these dioxygenases as well as their potential role in spider mite xenobiotic metabolism remains however poorly understood. In another study, Ahn et al. (2014) showed by in-depth phylogenetic analysis that UGT genes were lost early in chelicerate evolution, but then regained through HGT from bacteria in the phytophagous Tetranychidae lineage. After the transfer event, T. urticae proliferated the laterally acquired UGT gene to 80 paralogues in its genome. Ahn et al. (2014) also indicated that these horizontally transferred UGTs might play a role in the xenobiotic metabolism by identifying a substantial transcriptional response when exposed to different plant diets as well as between pesticide resistant and pesticide susceptible mite strains.

1.7. GENERAL OUTLINE OF THIS THESIS In this thesis, I aimed at further unravelling two major research questions: (1) did plant-feeding spider mites indeed co-opt laterally acquired genes in their xenobiotic metabolism, and (2) how do spider mites adapt to new host plants on the molecular level and do these molecular changes show signs of pesticide

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TABLE 1.2. Set of genes in the T. urticae genome of which the best BLAST hits were of bacterial or fungal origin. Description TeturID Cobalamin-independent methionine synthase tetur16g00990

Levanase tetur29g01230 tetur29g01280

Carotenoid cyclase/synthase tetur01g11260 tetur11g04840

Carotenoid desaturase tetur01g11270 tetur11g04820 tetur11g04810

Intradiol ring-cleaving dioxygenase tetur01g00490 tetur12g04671 tetur04g00150 tetur13g04550 tetur04g08620 tetur19g02300 tetur06g00450 tetur19g03360 tetur06g00460 tetur20g01160 tetur07g02040 tetur20g01790 tetur07g05930 tetur28g01250 tetur07g05940 tetur44g00140 tetur07g06570

Cyanase tetur28g02430 Grbic et al. (2011) identified these potentially horizontally transferred genes by means of an automated bioinformatics pipeline. Tetranychus urticae gene IDs can be accessed at http://bioinformatics.psb.ugent.be/orcae/overview/Tetur

resistance. Throughout my thesis, I used T. urticae as a model organism and took its recently sequenced genome as a main starting point. In Chapter 2, the putatively horizontally transferred T. urticae cyanase gene (TABLE 1.2) was heterologously expressed to verify if it still codes for a functional protein. Its evolutionary history and its potential role in detoxifying cyanogenic plant defenses were more closely examined. In Chapter 3, transcriptome analysis of spider mite adaptation to plant cyanogenesis revealed a novel laterally acquired gene (Tu-CAS). Recombinant expression was used to investigate whether the mite enzyme can directly detoxify cyanide, the toxin produced by cyanogenic plants. In Chapter 4, the T. urticae transcriptional sig- natures associated with phytochemical exposure and pesticide resistance were dissected and used to further explore the spider mite xenobiotic metabolism. In Chapter 5, T. urticae was adapted to tomato. The transcriptomic changes affili- ated with the genetic adaptation were identified and analyzed in light of the rel-

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ative roles of detoxification versus manipulation of plant defenses upon host plant adaptation. In Chapter 6, the question whether HGT has contributed to the evolution of arthropod herbivory is reviewed.

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Hilbrant, M., Damen, W.G.M. and Mcgregor, A.P. (2012) Evolutionary crossroads in develop- mental biology: the spider Parasteatoda tepidariorum. Development, 139, 2655-2662. Hill, C.A. (2010) Genome analysis of major tick and mite vectors of human plant pathogens. Available online: http://www.genome.gov/Pages/Research/DER/PathogensandVectors/Tick_and_ Mite_Genomes_Cluster_White_Paper_12Jan2011.pdf. Hill, R.L. and Odonnell, D.J. (1991) The Host Range of Tetranychus lintearius (Acarina, Tetranychidae). Experimental & Applied Acarology, 11, 253-269. Hotopp, J.C.D. (2011) Horizontal gene transfer between bacteria and animals. Trends in Genetics, 27, 157-163. Hotopp, J.C.D., Clark, M.E., Oliveira, D.C.S.G., Foster, J.M., Fischer, P., Munoz Torres, M.C., Giebel, J.D., Kumar, N., Ishmael, N., Wang, S., Ingram, J., Nene, R.V., Shepard, J., Tomkins, J., Richards, S., Spiro, D.J., Ghedin, E., Slatko, B.E., Tettelin, H. and Werren, J.H. (2007) Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science, 317, 1753-1756. Howe, G.A. and Jander, G. (2008) Plant immunity to insect herbivores. Annual Review of Plant Biology. pp. 41-66. Hoy, M.A. (2009) The predatory mite Metaseiulus occidentalis: mitey small and mitey large genomes. Bioessays, 31, 581-590. James, B.D. (1988) Tetranychid mites on cassava in Sierra Leone. Insect Science and Its Application, 9, 243-247. Jeppson, L.R., Keifer, H.H. and Baker, E.W. (1975) Mites injurious to Economic Plants. University of California Press, Berkely, Los Angeles. Joussen, N., Agnolet, S., Lorenz, S., Schoene, S.E., Ellinger, R., Schneider, B. and Heckel, D.G. (2012) Resistance of Australian Helicoverpa armigera to fenvalerate is due to the chimeric P450 enzyme CYP337B3. Proceedings of the National Academy of Sciences of the United States of America, 109, 15206-15211. Kant, M.R., Sabelis, M.W., Haring, M.A. and Schuurink, R.C. (2008) Intraspecific variation in a generalist herbivore accounts for differential induction and impact of host plant defences. Proceedings of the Royal Society B-Biological Sciences, 275, 443-452. Keeling, C.I., Yuen, M.M.S., Liao, N.Y., Docking, T.R., Chan, S.K., Taylor, G.A., Palmquist, D.L., Jackman, S.D., Anh, N., Li, M., Henderson, H., Janes, J.K., Zhao, Y., Pandoh, P., Moore, R., Sperling, F.a.H., Huber, D.P.W., Birol, I., Jones, S.J.M. and Bohlmann, J. (2013) Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest. Genome Biology, 14. Kirsch, R., Gramzow, L., Theissen, G., Siegfried, B.D., Ffrench-Constant, R.H., Heckel, D.G. and Pauchet, Y. (2014) Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: Key events in the evolution of herbivory in beetles. Insect Biochemistry and Molecular Biology, 52, 33-50. Knowles, C.O. (1997) Mechanisms of resistance to acaricides. Molecular Mechanisms of resistance to agrochemicals. Springer Berlin Heidelberg, 57-77. Koonin, E.V., Makarova, K.S. and Aravind, L. (2001) Horizontal gene transfer in prokaryotes: Quantification and classification. Annual Review of Microbiology, 55, 709-742. Krantz, G.W. and Lindquist, E.E. (1979) Evolution of Phytophagous Mites (Acari). Annual Review of Entomology, 24, 121-158. Krieger, R.I., Feeny, P.P. and Wilkinson, C.F. (1971) Detoxication enzymes in the guts of caterpillars: an evolutionary answer to plant defenses? Science (New York, N.Y.), 172, 579-581.

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