T. Van Leeuwen

The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance

The cys-loop ligand-gated ion channel (cysLGIC) super family of Tetranychus urticae, the two-spotted spider mite, represents the largest arthropod cysLGIC super family described to date and the first characterised one within the group of chelicerates. Genome annotation, phylogenetic analysis and comparison of the cysLGIC subunits with their counterparts in insects reveals that the T. urticae genome encodes for a high number of glutamate- and histamine-gated chloride channel genes (GluCl and HisCl) compared to insects. Three orthologues of the insect γ-aminobutyric acid (GABA)-gated chloride channel gene Rdl were detected. Other cysLGIC groups, such as the nAChR subunits, are more conserved and have clear insect orthologues. Members of cysLGIC family mediate endogenous chemical neurotransmission and they are prime targets of insecticides. Implications for toxicology associated with the identity and specific features of T. urticae family members are discussed. We further reveal the accumulation of known and novel mutations in different GluCl channel subunits (Tu_GluCl1 and Tu_GluCl3) associated with abamectin resistance in T. urticae, and provide genetic evidence for their causality. Our study provides useful toxicological insights for the exploration of the T. urticae cysLGIC subunits as putative molecular targets for current and future chemical control strategies.

Identification of pyrethroid resistance associated mutations in the para sodium channel of the two‐spotted spider mite Tetranychus urticae (Acari: Tetranychidae)

We investigated pyrethroid resistance mechanisms in Tetranychus urticae strains from Greece. Combined bioassay, biochemical and synergistic data indicated that although P450 mono‐oxygenase activities were associated with the trait, target site insensitivity was the major resistance component. A 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III with an unknown role. Three transcripts were identified corresponding to the k and l alternative exons. The mode of inheritance of resistance was confirmed as incompletely recessive, which is consistent with a target site mechanism for pyrethroids.

Resistance mechanisms to mitochondrial electron transport inhibitors in a field-collected strain of Tetranychus urticae Koch (Acari: Tetranychidae)

A Belgian field strain (MR-VP) of Tetranychus urticae (Koch) (Acari: Tetranychidae) exhibits different levels of resistance to four frequently used METI (mitochondrial electron transport inhibitor)-acaricides, i.e. tebufenpyrad, fenpyroximate, pyridaben and fenazaquin. Resistance factors for these compounds were 184, 1547, 5971 and 35, respectively. A 23.5-fold increase in 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity suggested that metabolic resistance through elevated levels of cytochrome P450 dependent monooxygenase-activity is a possible resistance mechanism.However, synergism studies with different metabolic inhibitors revealed some contrasting resistance mechanisms between the METI-acaricides. Tebufenpyrad resistance could only be synergized after pre-treatment with the monooxygenase inhibitor piperonyl butoxide (PBO), whereas pyridaben resistance was strongly synergized both by PBO and the esterase inhibitor S,S,S-tributylphosphorotrithioate (DEF). Resistance levels to fenpyroximate could neither be suppressed by PBO nor by DEF. Although METI-acaricides are structurally related, these findings probably reflect a different role of esterases and mono-oxygenases in metabolic detoxification between these compounds. The overall lack of synergism by diethylmaleate (DEM) suggests that glutathione-S-transferases are not an important factor in resistance to METIs.Reciprocal crosses between susceptible females and resistant males showed no maternal effect, and resistance to METI-acaricides was inherited generally as a dominant trait. Backcrosses with F1 females revealed striking differences in the mode of inheritance. Although resistance to fenpyroximate and pyridaben was under monogenic control, resistance to tebufenpyrad was under control of more than one gene.

Parallel evolution of cytochrome b mediated bifenazate resistance in the citrus red mite Panonychus citri

Bifenazate is a recently developed acaricide that is mainly used to control spider mites on a variety of crops. Although first thought to be a neurotoxin, genetic evidence obtained from bifenazate resistant Tetranychus urticae strains suggested an alternative mode of action as a Qo pocket inhibitor of the mitochondrial complex III. In this study, we reveal how bifenazate resistance in strains of Panonychus citri is maternally inherited and can confer cross‐resistance to the known Qo inhibitor acequinocyl. The mitochondrial genome of P. citri was sequenced and Qo pocket mutations were shown to be linked with the resistant trait. Parallel evolution of cytochrome b mediated bifenazate resistance corroborates the alternative mode of action and yet again illustrates that care should be taken when employing Qo inhibitors as crop protection compounds.

A horizontally transferred cyanase gene in the spider mite Tetranychus urticae is involved in cyanate metabolism and is differentially expressed upon host plant change

The genome of the phytophagous two-spotted spider mite Tetranychus urticae was recently sequenced, representing the first complete chelicerate genome, but also the first genome of a highly polyphagous agricultural pest. Genome analysis revealed the presence of an unexpected high number of cases of putative horizontal gene transfers, including a gene that encodes a cyanase or cyanate lyase. In this study we show by recombinant expression that the T. urticae cyanase remained functionally active after horizontal gene transfer and has a high affinity for cyanate. Cyanases were also detected in other plant parasitic spider mites species such as Tetranychus evansi and Panonychus citri, suggesting that an ancient gene transfer occurred before the diversification within the Tetranychidae family. To investigate the potential role of cyanase in the evolution of plant parasitic spider mites, we studied cyanase expression patterns in T. urticae in relation to host plant range and cyanogenesis, a common plant defense mechanism. Spider mites can alter cyanase expression levels after transfer to several new host plants, including the cyanogenic Phaseolus lunatus. However, the role of cyanase is probably not restricted to cyanide response, but likely to the plant nutritional quality as a whole. We finally discuss potential interactions between cyanase activity and pyrimidine and amino acid synthesis.

Wolbachia induces strong cytoplasmic incompatibility in the predatory bug Macrolophus pygmaeus

Macrolophus pygmaeus is a heteropteran predator that is widely used in European glasshouses for the biological control of whiteflies, aphids, thrips and spider mites. We have demonstrated that the insect is infected with the endosymbiotic bacterium Wolbachia pipientis. Several gene fragments of the endosymbiont were sequenced and subsequently used for phylogenetic analysis, revealing that it belongs to the Wolbachia supergroup B. The endosymbiont was visualized within the ovarioles using immunolocalization. Tetracycline treatments were used to cure M. pygmaeus from its infection. Although a completely cured line could not be obtained by this approach, the application of a constant antibiotic pressure over 13 generations resulted in a line with a significantly reduced Wolbachia concentration. Crosses performed with this tetracycline‐treated line revealed that the endosymbiont causes severe cytoplasmic incompatibility. This is the first report of a reproductive effect induced by Wolbachia in an economically important heteropteran predator that may have vital implications for its commercial production and use in biological control.

A heterogeneity based clustering heuristic for mobile ad hoc networks

An ad hoc network is an autonomous system of heterogeneous, mobile nodes that communicate with each other over wireless links. Routing protocols for these networks are inherently based on broadcasting control information and are therefore very bandwidth consuming. In order to limit the amount of routing information that has to be stored and maintained by the individual nodes, the technique of clustering is used. The network is partitioned into nonoverlapping sub networks, referred to as clusters, and one cluster node, the clusterhead, will take a leading role in the dissemination of control information. In this paper we approach the problem of finding an optimal partition that explicitly takes into account the heterogeneity of the network, as an integer linear programming (ILP) problem. In a second phase we have developed a new heuristic that approximates our ILP solution that is used in our clustering algorithm. It is shown that this heuristic tends to be more stable than existing clustering techniques that are solely based on ID number and/or connectivity and that do not take into account the heterogeneity of the network.

QoS-enabled Internet-on-train network architecture: inter-working by MMP-SCTP versus MIP

Internet-on-the-train is a rising concept in the last few years. Several trials in different countries have proved the feasibility of offering Internet access to train commuters, but none of them combines broadband access, scalability, seamless handover and quality of service guarantees in one solution. In this paper, we propose a new architecture to satisfy these needs. Using real handover measurement data of several common broadband wireless technologies, we compare two possible inter-working mobility solutions: Mobile Multi-Path SCTP (MMP-SCTP) and Mobile IP (MIP).

Preserving Streaming Video Quality in Mobile Wireless LAN networks

Handoffs between access points in a distributed IEEE 802.11 wireless LAN network for vehicular users can be a source of considerable packet loss. This severely degrades the visual quality of streaming video applications. In this paper we introduce the architecture of an Ethernet soft switch, which tries to preserve the quality of high bit rate streaming video upon each handoff, even if only limited resources are available access points. We have evaluated its performance in a test bed implementation for MPEG-2, MPEG-4 and H.264 video-on-demand and have compared it to the standard IEEE 802.11F-IAPP approach