Insect Biochemistry and Molecular Biology 45 (2014) 89E110

Insect Biochemistry and Molecular Biology 45 (2014) 89e110

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Insect Biochemistry and Molecular Biology

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Review The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistanceq

Wannes Dermauw a,**, Thomas Van Leeuwen a,b,* a Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium b Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands article info abstract

Article history: About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white Received 4 November 2013 gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is Received in revised form now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC 6 November 2013 proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large Accepted 6 November 2013 diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and Keywords: exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a cheli- Traffic ATPases Primary carriers cerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven Allelochemicals arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest Multidrug resistance conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE Detoxification and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and Phase III discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant alle- Arthropoda lochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in P-gp xenobiotic resistance in arthropods is historically not well documented, but an increasing number of MDR studies using unbiased differential gene expression analysis now points to their importance. We give an MRP overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also BCRP SUR recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the fi Bt toxins enormous interest in Bt toxicology in transgenic crops, such ndings will provide an impetus to further reveal the role of ABC transporters in arthropods. Ó 2014 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Introduction Hediger et al., 2013). Among the latter, the ABC (ATP-binding cassette)1 protein family is one of the largest transporter families All cells and organelles are separated from the external milieu by and is present in all kingdoms of life. The majority of these ABC lipid membranes and need transporters to trafﬁc a wide diversity of proteins function as primary-active transporters, requiring the compounds across these membranes (Higgins, 1992). The impor- binding and hydrolysis of ATP to transport substrates across lipid tance of membrane transport is illustrated by the fact that in membranes. A functional ABC transporter, previously also referred to Escherichia coli and humans about 10% and 4% of the total number of as trafﬁc ATPase, consists of two cytosolic nucleotide-binding do- genes encode transport-related proteins (Blattner et al., 1997; mains (NBDs) that bind and hydrolyze ATP, and two integral transmembrane domains (TMDs) (Fig. 1A). The NBD domain contains several conserved sequences like a Walker A and Walker B motif, Q-

q loop, H-motif and the ABC-signature motif (LSGGQ-motif). The This is an open-access article distributed under the terms of the Creative transmembrane domains consist of 5e6 transmembrane helices and Commons Attribution-NonCommercial-No Derivative Works License, which per- mits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. * Corresponding author. Laboratory of Agrozoology, Department of Crop Protec- 1 ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, trans- tion, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B- membrane domain; FT, full transporter; HT, half transporter; BBB, bloodebrain 9000 Ghent, Belgium. Tel.: þ32 92646154. barrier; MRPs, multidrug-resistance associated proteins; MDR, multidrug-resis- ** Corresponding author. Tel.: þ32 92646155. tance protein, P-gp, P-glycoprotein; SUR, sulfonylurea receptor; TMR, tetrame- E-mail addresses: [email protected] (W. Dermauw), thomas. thylrosamine; Trp, Tryptophan; ML, macrocyclic lactone; OP, organophosphates; [email protected] (T. Van Leeuwen). BPU, benzoylphenylureas.

0965-1748/$ e see front matter Ó 2014 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibmb.2013.11.001 90 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110

A 2 TMD1 TMD2 OUT 2

2 3 4 5 6 8 9 10 11 12

IN NH2 NBD NBD COOH

B step I step II step III step IV

OUT

NBD

D ATP

D IN D

B ATP B

N ATP

Fig. 1. ABC full transporter structure and the ATP-switch model for the transport cycle of an ABC transporter (exporter-type) A) Typical structure of an ABC full transporter containing two TMDs, TMD1 (blue) and TMD2 (yellow) each containing 6 transmembrane (TM) segments, and two NBDs, NBD1 (red) and NBD2 (green). In the case of “long” MRPs an additional TMD (TMD0) is present at the N-terminus (Deeley et al., 2006). ABC half transporters have only one TMD and one NBD and need to homo or heterodimerize to form a functional unit. B) The ATP-switch mechanism (Higgins and Linton, 2004; Linton and Higgins, 2007). The transport cycle is started by binding of a substrate (purple cross) to a high- affinity pocket formed by the TMDs (blue and yellow pentagon). Subsequently, a conformational change is transmitted to the NBDs (green and red), facilitating ATP (orange oval) binding and closed NBD-dimer formation. The closed NBD dimer induces on its turn a major conformational change in the TMDs, with TMDs rotating and opening toward the outside, initiating substrate translocation (step II). ATP hydrolysis initiates dissolution of the closed NBD dimer, resulting in further conformational changes in the TMDs (step III). Finally, phosphate and ADP release restores the transporter to the open NBD-dimer conformation (step IV). provide substrate specificity. The four domains of a functional resistance of cancer cells against chemotherapeutics: the multidrug- transporter can be present in one protein (Full Transporter, FT) or resistance proteins (MDRs) or P-glycoproteins (P-gps) belonging to spread over multiple proteins, for example one NBD and TMD per the ABCB subfamily, the multidrug-resistance associated proteins protein (half transporter, HT). In the case of the latter, ABC proteins (MRPs) from the ABCC subfamily and the breast cancer resistance need to homo or heterodimerize to form a functional ABC trans- protein (BCRP, ABCG subfamily member). The P-gp ABCB1 was in fact porter (Higgins, 1992; Higgins and Linton, 2004; Rees et al., 2009). identified as the first ABC transporter to be overexpressed in Based on the sequence similarity of the NBDs, the ABC protein family multidrug resistant tumor cell lines (Kartner et al., 1985; Riordan has been divided into eight subfamilies, denoted by the letters AeH et al., 1985). Compared to bacterial, nematode and human ABC (Fig. 2). Of particular note, the ABCH subfamily has at present only transporters, the knowledge about arthropod ABC transporters is been identified in arthropod genomes and in zebrafish (Dean et al. still limited. At present, the ABC families of seven arthropod species, 2001; Popovic et al., 2010). Eukaryotic ABC transporters mediate spanning more than 400 arthropod ABC proteins, have been studied the efflux of compounds from the cytoplasm to the outside of the cell in detail (Broehan et al. 2013; Dean et al., 2001; Dermauw et al., or into organelles, while bacterial ABC transporters can also facilitate 2013a; Liu et al., 2011; Roth et al., 2003; Sturm et al., 2009; Xie import of substances. However, not all ABC proteins do play a role in et al., 2012). Nevertheless, the precise function of only a few transport. In the human ABC protein family, for example, ABC pro- arthropod transporters has been unveiled. Drosophila melanogaster teins acting as ion channel, receptor or with a role in translation, white and its insect orthologues are probably the most thoroughly have been characterized (Dean et al., 2001; Rees et al., 2009). characterized ABC transporters and are involved in the uptake of Currently, the ATP-switch mechanism is the favored model for pigment precursors in the developing eye (Ewart and Howells,1998). the transport cycle of ABC transporters (George and Jones, 2012; We performed a thorough phylogenetic analysis with a dataset Higgins and Linton, 2004; Linton and Higgins, 2007). In this model, of well-annotated arthropod and human ABC transporters to infer the transport cycle is started by binding of substrates to the TMDs. orthologous relationships, which in turn can suggest the role and Subsequently a conformational change is transmitted to the NBDs, function of arthropod proteins. We further focus on biochemical facilitating ATP binding and closed NBD-dimer formation. The closed and molecular methods that can be deployed to study arthropod NBD dimer in turn induces a major conformational change in the ABC transporters, and give an overview of their documented role in TMDs, with TMDs rotating and opening toward the outside, initi- xenobiotic transport and resistance. ating substrate translocation. In a final step, ATP is hydrolyzed to initiate transition of the closed NBD dimer to the open NBD dimer 2. Comparison of ABC subfamilies in arthropods and to return the transporter to its basal state (Fig. 1B). ABC- transporters traffic an array of substrates covering amino-acids, Despite the fact that more than 60 arthropod genomes have sugars, lipids, inorganic ions, polysaccharides, metals, peptides, toxic been sequenced, the ABC protein family was annotated and studied metabolites and drugs. Because of their ability to transport drugs, in detail in only seven species: the dipterans D. melanogaster and many human ABC members are also important players in multidrug Anopheles gambiae, the honeybee Apis mellifera, the silk moth W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 91

Fig. 2. A schematic tree depicting the phylogenetic relationships between ABC subfamilies. NBDs of ABC proteins belonging to different subfamilies as deﬁned by Dermauw et al. (2013a) were used in a ML analysis. Similar to previous studies (e.g. Liu et al., 2011) C-terminal NBDs of the ABCC subfamily clustered together with NBDs of the ABCB subfamily.

Bombyx mori, the flour beetle Tribolium castaneum, the spider mite 2.1. The ABCA family Tetranychus urticae and the waterflea Daphnia pulex (Broehan et al., 2013; Dean et al., 2001; Dermauw et al., 2013a; Liu et al., 2011; Roth Phylogenetic analysis of the ABCA subfamily revealed a clear et al., 2003; Sturm et al., 2009; Xie et al., 2012)(Table 1). Among orthologous relationship between D. melanogaster CG31731, these species, T. urticae has the largest number of ABC genes (103) A. gambiae AGAP001523, T. castaneum TcABCA-7 and TcABCA-UA, followed by T. castaneum (73) and D. pulex (65), while A. mellifera A. mellifera AmABCA2 and B. mori BGIBMGA004187/ has only 41 ABC genes (Table 1). The large number of ABC genes in BGIBMGA004188 (Supplementary Fig. 1, Table 2). This group of T. urticae is mainly due to lineage-specific expansions of ABCC, insect ABCA FTs cluster together with high bootstrap support with 7 ABCG and ABCH genes, while in T. castaneum and D. pulex lineage- ABCA FTs of T. urticae. Except for CG31731, reported to be down- specific expansions of ABCC and ABCH genes have been docu- regulated in the salivary glands of an E93 mutant of D. melanogaster mented (Broehan et al., 2013; Dermauw et al., 2013a; Sturm et al., (Dutta, 2008), no information is available on the function of these 2009)(Table 1). In order to infer orthologous relationships be- arthropod ABCA transporters. Other orthologous relationships tween arthropod ABC proteins, we performed a maximum likeli- were found between A. gambiae AGAP010416, A. mellifera hood analysis for each ABC subclass using complete ABC protein AmABCA3, T. castaneum TcABCA-6A, B. mori BGIBMGA012789 and sequences from Homo sapiens and the before mentioned arthropod D. pulex Dappudraft_346971 and human ABCA5, 6, 8e10 and be- species (for details see Supplementary Text). tween A. gambiae AGAP010582, D. melanogaster CG34120,

Table 1 Gene numbers in ABC subfamilies of H. sapiens and seven arthropod species.a

ABC subfamily H. sapiens D. melanogaster A. gambiae T. castaneum A. melliferab B. moric D. pulex T. urticae

A12109103949 BFT 4 4 1 2 1 5 2 2 BHT 7 4 4 4 4 4 5 2 C 12 14 13 35 9 15 7 39 D42222232 E11111111 F33333343 G 5 15 16 13 15 13 24 23 H0333331522 TOTAL 48 56 52 73 41 55 65 103

a Numbers were derived from Sturm et al. (2009), Liu et al. (2011), Xie et al. (2012), Broehan et al. (2013) and Dermauw et al. (2013a). b AmABCB5 and AmABCB6 from Liu et al. (2011) were not taken into account as they were incomplete (less than 270 AA), their respective genes were located on unplaced scaffolds and had best BLASTp hits (E-value e 100) with ABC transporters of bacteria. c The number of B. mori ABC genes for each ABC subfamily represents the highest number of B. mori genes found for each ABC subfamily in either Liu et al. (2011) or Xie et al. (2012), some B. mori ABC genes might be incomplete and might possibly be combined into one gene (e.g. BGIBMGA007792/BGIBMGA007793 (Atsumi et al., 2012), BGIBMGA007784/BGIBMGA007785 and BGIBMGA4187/BGIBMGA04188). Table 2 92 Orthologous relationships between ABC proteins of Arthropoda and H. sapiens. e

Dma Ag Tc b Am Bm Dp Tu Hs Function? Reference

ABCA e AGAP010416 TcABCA-6A AmABCA3 BGIBMGA012789 Dappudraft_346971 e hABCA5/6/8-10 Lipid transport d? Wenzel et al., 2007, CG34120 AGAP010582 e AmABCA1 BGIBMGA009503 e tetur27g01890 hABCA12/13 ABCA12 is a keratinocyte lipid transporter d; Liu and Lehmann 2008; Akiyama, GAL4 changes CG34120 expression, 2013 independent of UAS c CG31731 AGAP001523 TcABCA-7A AmABCA2 BGIBMGA004187 e (7) e CG31731 is downregulated in E93 mutants Dutta, 2008 TcABCA-UA BGIBMGA004188 ABCB HT CG7955 AGAP006364 TcABCB-5A AmABCB1 BGIBMGA012743 Dappudraft_347270 tetur32g01330 hABCB7 Mitochondrial ABC transporters with roles in Zutz et al., 2009; Sooksa-Nguan CG4225 AGAP002278 TcABCB-6A AmABCB2 BGIBMGA005473 Dappudraft_347268 e hABCB6 biogenesis of cytosolic ironesulfur clusters, et al., 2009; Bariami et al., 2012; CG1824AGAP006273 TcABCB-7A AmABCB7 BGIBMGA004142 Dappudraft_347266 tetur17g02000 hABCB8 heme biosynthesis, iron homeostasis, Broehan et al., 2013 d 89 (2014) 45 Biology Molecular and Biochemistry Insect / Leeuwen Van T. Dermauw, W. CG3156 AGAP002717 TcABCB-4A AmABCB3 BGIBMGA008523 Dappudraft_347275 e hABCB10 protection against oxidative stress ; CG4255 is Dappudraft_347276 involved in cadmium tolerance; RNAi silencing of TcABCB-5A results in severe developmental defects during pupation and abortive pupal- adult molting; role in pesticide resistance? ABCC sur AGAP009799 e AmABCC2 BGIBMGA006882 Dappudraft_442500 tetur11g05990 hABBC8/9 Subunit of the ATP-sensitive potassium channel Nasonkin et al., 1999 (SUR1/2) c c CG6214 AGAP009835 TcABCC-9A AmABCC9 (3) Dappudraft_347281 (23) hABCC1/2/3/6 Drosophila CG6214 is capable of transporting Tarnay et al., 2004; Szeri et al., 2009 (dMRP) AGAP008437 (MRP1, 2, 3 and 6) several substrates of its human orthologs CG7806 AGAP007917 TcABCC-4A AmABCC5 BGIBMGA010636 Dappudraft_347323 tetur03g07840 hABCC10 (MRP7) Transport of taxanes d; CG7806 is upregulated Beckstead et al., 2007; Hopper- 1.56-fold in D. melanogaster upon exposure to Borge et al., 2009 ecdysone; conserved function in metazoa? ABCD CG2316 AGAP002071 TcABCD-6A AmABCD1 BGIBMGA004616 Dappudraft_347330 tetur05g06640 hABCD1/2 Import of long branched chain acyl-coA into Morita and Imanaka, 2012 d CG12703AGAP000440 TcABCD-9A AmABCD2 BGIBMGA012688 Dappudraft_347326 tetur35g01360 hABCD3 peroxisome ABCE CG5651 AGAP002182 TcABCE-3A AmABCE1 BGIBMGA010129 Dappudraft_189585 tetur30g0140 hABCE1 Essential role in ribosome biogenesis and Andersen and Leevers, 2007 (pixie) translation regulation ABCF d CG1703 AGAP012249 TcABCF-2A AmABCF1 BGIBMGA007869 Dappudraft_347357 tetur29g00620 hABCF1 (ABC50) Essential role in translation regulation Paytubi et al., 2009 Dappudraft_347363 CG9281AGAP002693 TcABCF-5A AmABCF3 BGIBMGA002004 Dappudraft_304799 tetur20g02610 hABCF2 CG9330 AGAP012005 TcABCF-9A AmABCF2 BGIBMGA006964 Dappudraft_347354 tetur32g00490 hABCF3 ABCG CG3327 e TcABCG-8A AmABCG1 e Dappudraft_347416 tetur17g02510 e 20E induced ABC transporter, injection of Hock et al., 2000; Broehan et al., (E23) TcABCG-8A dsRNA results in molting defects 2013, and developmental arrest c (9)c e Best characterized ABC transporter: transport of Zhang and Odenwald, 1995; Ewart

White AGAP000553 TcABCG-9B AmABCG2 BGIBMGA002922 (9) e

eye pigment precursors, role in courtship and Howells 1998; Borycz et al., 110 behavior, uptake of uric acid, transport of cyclic 2008; Evans et al., 2008; Komoto et GMP and transport of biogenic amines. al., 2009; Broehan et al., 2013 Brown AGAP007655 e AmABCG11 BGIBMGA002581 eeeTransport of eye pigment precursor; transport Ewart and Howells 1998, Borycz et AMABCG12 BGIBMGA002712 of biogenic amines al., 2008 Scarlet AGAP000506 TcABCG-9A AmABCG10 BGIBMGA002924 Dappudraft_347393 ee Transport of eye pigment precursor;transport of Ewart and Howells 1998, Borycz et AGAP001333 biogenic amines al., 2008, Tatematsu et al., 2011 d CG11069AGAP002051 TcABCG-9D AmABCG15 BGIBMGA000472 Dappudraft_300887 tetur01g16290 hABCG5 Role in biliary excretion Graf et al., 2003 CG31121AGAP002050 TcABCG-9C AmABCG13 BGIBMGA000220 Dappudraft_258299 tetur01g16280 hABCG8 Role in biliary excretion d Graf et al., 2003 Atet AGAP001858 TcABCG-4D AmABCG3 BGIBMGA005094 Dappudraft_347444 e hABCG1/4 ABC transporter expressed in trachea of Kuwana et al., 1996 D. melanogaster CG3164 AGAP009850 TcABCG-4C AmABCG5 BGIBMGA005202 eeeCG3164 knockdown line results in lethal Ohmann 2012, Broehan et al., 2013, phenotype in pupa; essential in D. melanogaster Liu et al., 2011 lipid metabolism; transport of lipids from epidermal cells to the cuticle in T. castaneum; injection of TcABCG-4C dsRNA in young females W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 93

H. A. mellifera AmABCA1, B. mori BGIBMGA009503, T. urticae tetur27g01890 and human ABCA12 and 13 (see Supplementary Fig. 1). Little is known about the function of human ABCA5, 6, 8e 10 but several studies have suggested roles of these transporters in lipid transport (Wenzel et al., 2007). Human ABCA12 on the other hand has been thoroughly characterized and is known as a keratinocyte lipid transporter (Akiyama, 2013). The role of the arthropod orthologues of these human ABCAs is unknown, but they might play a role in lipid transport. Finally, we also identified a well-supported group of insect-specific ABCA proteins, containing lineage-specific expansions in B. mori, T. castaneum, D. melanogaster Liu et al., 2011 Buchmann et al., 2007, Liu2011 et al., Qin et al., 2005 Mummery-Widmer et al., 2009, Zhang et al., 2009, Broehan2013 et al., e Etter et al., 2005, Liu et al., 2011 Pedra et al., 2004, Ellis2010 and Carney Liu et al., 2011 and A. gambiae (see Supplementary Fig. 1). To date, only one study has investigated the function of ABC proteins in this insect-specific ABCA group. Injection of dsRNA targeting TcABCA-9A or TcABCA-9B ; 20E of T. castaneum resulted in 30% mortality during the pupaeadult molt and pupae and pharate adults showed severe wing defects B. mori B. mori B. mori D. melanogaster and elytra shortening (Broehan et al., 2013). Future studies might B. mori ies after cold

fl confirm that this group of insect-specific ABCAs is involved in D. melanogaster onding ABC family. (wing) development.

male heads after mating 2.2. The ABCB family B. mori . The ABCB subfamily consists of half and full transporters (HTs and FTs, respectively, see above). The phylogenetic analysis of H. sapiens

, ABCB FTs, also referred to as P-gps, assigned H. sapiens, D. pulex, reduces number of eggs and20E eggs induced fail ABC to transporter hatch; in induced ABC transporter in Insertion of a engineered transposableelement P- (EP) upstream of CG17646triglyceride altered content the of Upregulated in adult fruit Downregulation of CG5853 following neuronal AP1 overexpression; 20E induced ABC transporter in hardening Similar to CG3164/TcABCG-4C ? CG31689 is overtranscribed in a DDT-resistant line; downregulated in D. melanogaster 20E induced ABC transporter in Hs fi , T. urticae and insects into separate clades, con rming an earlier hypothesis by Sturm et al. (2009) that this subfamily diversified through lineage-specific duplications (Fig. 3A). Within the insect T. urticae

, ABCB FT clade, 2 main groups can be observed. In the ﬁrst Tu

, Drosophila mdr50 clusters with TcABCB-3A, TcABCB-3B and BGIBMGA007494, while in the second Drosophila mdr65, CG10226 and mdr49 cluster with A. gambiae AGAP005639, A. mellifera D. pulex , AmABCB4 and B. mori BGIBMGA009452, BGIBMGA000724, Dp , BGIBMGA000725 and BGIBMGA011228 (Fig. 3A). The ABCB FTs in D. melanogaster have been extensively studied as mdr65 is

B. mori ﬂ , required for chemical protection of the fruit y brain by creating a

Bm bloodebrain barrier (BBB) (Mayer et al., 2009) and mdr49 plays an , essential role in germ cell migration (Ricardo and Lehmann, 2009). Furthermore, it was demonstrated that exposure of

ve out of seven arthropod species and 2) be clustered with a bootstrap value of at least 80%; one-to-one orthologues in arthropods and ﬂ

ﬁ D. melanogaster ies to polycyclic aromatic hydrocarbons led to an A. mellifera , induced ABCB FT/P-gp expression (Vaché et al., 2006, 2007).

Am ’ , Chahine and O Donnell (2009) revealed that D. melanogaster adult eee eee eee eee eee eee ﬂies raised on a diet containing 0.1 mM of the antifolate drug methotrexate, showed a signiﬁcant increased expression of mdr49, mdr50 and to a lesser extent mdr65, in both the Malpi- T. castaneum ve insect species or

, ghian tubules and the gut. A recent study reported the interaction ﬁ Tc

, between P-gps and polyglutamine mediated pathogenesis in D. melanogaster. Using a D. melanogaster transgenic strain ee ee expressing repeats of glutamine, it was demonstrated that

A. gambiae increased ABCB FT/P-gp levels reduces eye degeneration, while ,

Ag reduction of ABCB FT/P-gp levels resulted in an increase of the , disease (Yadav and Tapadia, 2013). Interestingly, D. melanogaster AmABCG8 BGIBMGA010557and 20E inducedother ABC transporter in arthropod ABCB FTs have also frequently been linked to insecticide transport and/or resistance ((Buss and Callaghan, 2008), this study, see Section 4). Contrary to ABCB FTs, clear

D. melanogaster orthologous relationships were identiﬁed among arthropod ABCB , e TcABCG-4B AmABCG9 BGIBMGA005203 HTs (Fig. 3B). Human ABCB6e8 each have one ortholog in insects Dm

-naming. and D. pulex (Fig. 3B, Table 2) while one ortholog and two co- orthologues (Dappudraft_347275 and Dappudraft_347276) of hu-

c man ABCB10 were found in insects and D. pulex, respectively. Of

e particular note, two previously annotated ABCB HTs of A. mellifera (AmABCB5 and AmABCB6 (Liu et al., 2011)) were not included in

are underlined. our analysis as they were incomplete (less than 270 AA), their

Species abbreviations: Broehan et al. (2013) Number of orthologues that hasFunction been of found human in ortholog. thisOrthologous arthropod genes species, should gene 1) at names least of be orthologues present in can four be out found of in the phylogenetic analysis of the corresp respective genes were located on unplaced scaffolds and had best c CG17646 AGAP009464 TcABCG-4A AmABCG14 BGIBMGA010555 ABCH CG33970 AGAP002638 TcABCH-9B AmABCH3 BGIBMGA010726 CG5853 AGAP009463 TcABCG-4G AmABCG4 BGIBMGA005226 CG9990 AGAP003680 TcABCH-9CCG11147 AmABCH1 AGAP002060 BGIBMGA010760 TcABCH-9A AmABCH2 BGIBMGA010825 CG31689 AGAP008889 TcABCG-4F AmABCG6 CG4822 CG9664 (5) a e b d 100 sapiens BLASTp hits (E-value e ) with ABC transporters of bacteria, 94 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110

A B

Fig. 3. Phylogenetic analysis of ABCB full and half transporters of H. sapiens and seven arthropod species. A) ABCB full transporters, B) ABCB half transporters. For procedure and details of phylogenetic analysis see Supplementary Text. Color and shape codes are as follows: blue diamond, D. melanogaster, red diamond, A. gambiae, brown diamond, T. castaneum, green diamond, B. mori, yellow diamond, A. mellifera, purple square, D. pulex, gray triangle, T. urticae and black circle, H. sapiens. The scale bar represents in A) 0.2 and in B) 0.5 amino-acid substitutions per site.

potentially indicating contamination (data not shown). Contrary to lethality and 2) sterile females with ovaries failing to produce eggs. insects and D. pulex, only two orthologues of human ABCB HTs Nevertheless, based on the clear orthologous relationships be- (ABCB7 and ABCB8) were identified in T. urticae.Finally,no tween human and arthropod ABCB HTs and the fact that many of arthropod orthologues of human ABCB2 and ABCB3, both involved the arthropod ABCB HTs are predicted to be targeted to the in antigen processing (Trowsdale et al., 1990), and ABCB9 were mitochondria (predicted by TargetP 1.0 (Emanuelsson et al., 2007), identified in our evolutionary analysis (Fig. 3, Table 2). The group of data not shown), it is likely that arthropod ABCB HTs have similar evolutionary conserved ABCB HTs (orthologues of human ABCB6e roles as their counterparts in humans. 8 and 10) is known as mitochondrial ABC transporters with roles in the biogenesis of cytosolic ironesulfur clusters, heme biosynthesis, 2.3. The ABCC family iron homeostasis and protection against oxidative stress (Zutz et al., 2009). It was suggested recently that human ABCB6 is Human ABCC proteins are FTs with functionally diverse func- located in other cellular compartments than the mitochondria, but tions such as ion transport (e.g. human ABCC7, the cystic fibrosis the precise identity, substrate specificity and functionality has not transmembrane conductance regulator), cell-surface receptor ac- yet been well determined (Chavan et al., 2013). Hardly any func- tivity (e.g. human ABCC8 and 9, sulfonylurea receptors) and tional information is available on the function of arthropod translocation of a broad range of substrates like drugs, cyclic nu- orthologues of human ABCB HTs. Sooksa-Nguan et al. (2009) cleotides, endogenous/exogenous compounds and their gluta- showed through heterologous expression in Schizosacchar- thione conjugates (Dean et al., 2001; Keppler, 2011; Leslie et al., omyces pombe that the D. melanogaster ortholog of human ABCB6, 2005; Schinkel and Jonker, 2003). Because of their ability to CG4225 also known as D.melanogaster heavy metal tolerance extrude drugs with broad specificity, many ABCCs were historically factor 1 (DmHMT-1), could confer resistance against cadmium. named multidrug-resistance associated proteins (MRPs), with CG7955 was downregulated in D. melanogaster lines selected for “long” MRPs (human ABCC1-3, 6 and 10/MRP1e3, 6 and 7) having increased resistance to chill coma stress (Telonis-Scott et al., 2009). an extra N-terminal transmembrane spanning domain (TMD0) On the other hand, Broehan et al. (2013) found that injection of compared to “short” MRPs (human ABCC4, 5, 11 and 12/MRP4, 5, 8 TcABCB-5A dsRNA into pre-pupae of T. castaneum resulted in 1) and 9) (Deeley et al., 2006). Human MRP1 was the first MRP to be severe developmental defects during pupaeadult molt causing cloned and was identified in a multidrug resistant lung cancer cell W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 95

Fig. 4. Phylogenetic analysis of ABCC proteins of H. sapiens and seven arthropod species. For procedure and details of phylogenetic analysis see Supplementary Text. For color and shape codes see Fig. 3. The scale bar represents 0.5 amino-acid substitutions per site. One-to-one ABCC orthologues found in H. sapiens and seven arthropod species are shaded in grey, while examples of species-specific ABCC expansions are indicated with an arch. line (Cole et al., 1992; Cole, 2013). The various MRPs differ from P- Loe et al., 1998). In our phylogenetic analysis of the ABCC subfam- gps (ABCB FTs) by the ability to actively transport a broad range of ily, 23 T. urticae ABCC transporters of which the majority has a glucuronate, sulfate and glutathione (GSH)-conjugated organic TMD0,(Dermauw et al., 2013a) clustered with D. melanogaster anions. It has also been shown that several MRPs act in synergy CG6214, A. gambiae AGAP009835 and AGAP008437, A. mellifera with several phase II conjugating enzymes like the GSH-S- AmABCC9, T. castaneum TcABCC-9A, B. mori BGIBMGA010330, transferases (GSTs) and UDP-glycosyltransferases (UGTs) to confer BGIBMGA010331 and BGIBMGA010332 and a group of human resistance to electrophilic drugs and carcinogens (Leslie, 2012; Liu “long” MRPs (MRP1e3 and 6) (Fig. 4). Using recombinant expres- et al., 2012; Sau et al., 2010). Intriguingly, the two arthropod spe- sion in Sf9 cells and vesicular assays, it was reported that cies with the highest number of ABCCs, T. urticae and T. castaneum, D. melanogaster CG6214 (also named dMRP) shares the biochemical also have a clear expansion of the GST repertoire (Grbic et al., 2011; properties of its human orthologues, such as the transport of b- Shi et al., 2012). The T. urticae genome harbors an expansion of mu- estradiol 17-b-D-glucuronide, leukotriene C4 and calcein (Szeri class GSTs that are not present in insects and were until recently et al., 2009; Tarnay et al., 2004). It has also been shown that believed to be vertebrate specific(Grbic et al., 2011). Future studies alternative splicing results in multiple isoforms for D. melanogaster should reveal if there is a coordinated action between the high CG6214 and its orthologues in A. gambiae and Trichoplusia ni, which number of GSTs and the many ABCCs in T. urticae and T. castaneum. might generate functional diversity for this ABCC transporter Next to the transport of conjugated compounds, human MRPs can (Grailles et al., 2003; Labbé et al., 2011; Roth et al., 2003). also transport unconjugated compounds, including the chemo- Furthermore, Chahine and O’Donnell (2009, 2011) found that di- therapeutics vinblastine, etoposide, daunorubicin, and doxorubicin, etary exposure to the chemotherapeutic agent methotrexate or the in co-transport with GSH (Ballatori et al., 2009; Leslie et al., 2005; P450-monooxygenase inhibitor piperonylbutoxide resulted in an 96 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 upregulation of CG6214 in the Malpighian tubules of do not feed and the reduction in plant allelochemicals uptake may D. melanogaster. This group also discovered that reduction in the be linked to the downregulation of these ABCC genes (Bryon et al., expression of CG6214 was correlated with reduced secretion of the 2013). Next, it has been demonstrated that D. melanogaster chemotherapeutic daunorubicin (Chahine et al., 2012). Less is CG10505 contributes to metal homeostasis while D. melanogaster known about the other arthropod orthologues of human ABCC1e3 CG14709 regulates responsiveness to O2 deprivation and develop- and 6. Recently, Yoon et al. (2011) reported that the ortholog of ment under hypoxia (Huang and Haddad, 2007; Yepiskoposyan CG6214 in Pediculus humanus humanus (PhABCC4) was more highly et al., 2006). Recently, Shah et al. (2012) also showed that expressed after exposure to the pesticide ivermectin and that in- D. melanogaster CG4562 was upregulated in Cyp6g1 knockdown jection of PhABCC4 dsRNA altered the sensitivity of P. humanus to flies, suggestive of molecular interaction within a detoxification ivermectin (see also Section 4.2.2). network. The most enigmatic member from this large arthropod The evolutionary analysis also identified two clear orthologous ABCC-group clade is probably B. mori BGIBMGA007792e relationships between arthropod ABCCs and human ABCCs. One BGIBMGA007793 (these proteins were considered as separate ortholog of a human “long” MRP, human ABCC10/MRP7, was pre- transporters by Liu et al., 2011, but should be combined to form one sent in all arthropods included in our analysis (Fig. 4, Table 2). In full transporter (Atsumi et al., 2012)). An insertion in this B. mori contrast to human MRP1, not much is known about the function of transporter has been clearly linked to resistance against the Bacillus human ABCC10. This MRP is distinct from other human ABCCs in thuringiensis (Bt) Cry1A toxin but its precise role in Bt toxin mode of that it does not transport typical human MRP substrates like action is however unknown (see Section 4.2.2). Remarkably, no glutathione and sulfate conjugates. Unlike other MRPs, human clear orthologues of this lepidopteran transporter were detected in MRP7 is able to confer resistance to taxanes (Hopper-Borge et al., the other arthropod species included in our analysis (Fig. 4) and this 2009). Nevertheless, the presence of one-to-one orthologues might help explain why Cry1A toxins are exclusively toxic against with arthropods, suggests a more conserved function of this ABCC Lepidoptera (de Maagd et al., 2001). protein in humans and arthropods. The human sulfonylurea receptors (human ABCC8/SUR1 and human ABCC9/SUR2) clustered 2.4. The ABCD subfamily with D. melanogaster Sur, A. gambiae AGAP009799, B. mori BGIBMGA006882, A. mellifera AmABCC2, D. pulex Dappu- The ABCD subfamily consists of HTs that need to dimerize to draft_442500 and T. urticae tetur11g05990 (Fig. 4, Table 2). Sulfo- form a functional transporter. In humans they are involved in the nylurea receptors assemble with inwardly rectifying potassium import of long and branched chain acyl-CoA molecules into the (Kir) subunits, to form ATP-sensitive potassium channels (KATP). peroxisome (Morita and Imanaka, 2012). Both T. urticae and all These channels are involved in multiple physiological processes, insect species have two ABCD genes, while D. pulex and humans with roles in glucose homeostasis, ischemic protection and innate have three and four ABCD genes respectively (Table 2). The clear immunity (Akrouh et al., 2009; Eleftherianos et al., 2011). Sur- orthologous relationships between arthropod ABCDs and human prisingly, a clear ortholog of human SUR could not be identified in ABCD1/2 and ABCD3 suggest that their function is conserved the genome of T. castaneum (see also the OrthoDB group of (Fig. 5A, Table 2). arthropod SURs (EOG7HFCZ2) (Waterhouse et al., 2011)). In addition, we could not identify such orthologues in transcriptome data 2.5. The ABCE and ABCF family of other coleopterans (as determined by a tBLASTn search against coleopteran ESTs in NCBI and using human SUR1 and 2 as query), ABC proteins from the ABCE and ABCF family have 2 NBDs but suggesting that SUR was lost in the coleopteran lineage. However, lack TMDs and are involved in biological processes other than as orthologues of Kir are present in T. castaneum (as determined by transport. The ABCE1 protein is essential in all eukaryotes exam- a BLASTp search against the T. castaneum proteome using ined to date and plays a fundamental role in ribosome biogenesis D. melanogaster Irk1-PB as query) other ABC transporters might and translation regulation (Andersen and Leevers, 2007; Barthelme assemble with Kir to form a functional ATP-sensitive potassium et al., 2011). Contrary to human ABCE1 (also called Rli1p or OABP), channel. Although some reports suggest that SURs are the direct human ABCF1 (also called ABC50) is probably only involved in target of insecticidal benzoylphenylureas, a group of commercially translation regulation (Paytubi et al., 2009), while the precise role used chitin synthesis inhibitors, this theory is no longer favored of ABCF1 and ABCF2 is unclear. In our phylogenetic analysis, one (see Section 4.4.1). ortholog of human ABCE1, ABCF1e3 was found for each arthropod A number of ABCC proteins of D. pulex (Dappudraft_347292, species (Fig. 5B, Table 2). Based on these clear orthologous re- Dappudraft_347295 and Dappudraft_347548) and A. mellifera lationships, arthropod ABCE and ABCF proteins probably have an (AmABCC4) clustered with a group of “short” human MRPs (human analogous function as their human orthologues. An essential role of ABCC5, 11 and 12) that are characterized as transporters of (cyclic) ABCE and ABCF proteins was recently demonstrated for nucleotides/nucleosides and their analogs (Guo et al., 2003; Reid T. castaneum, where TcABCE-3A or TcABCF-2A dsRNA injection in et al., 2003)(Fig. 4). It has also been found that a SNP in the hu- penultimate larvae resulted in 100% mortality (Broehan et al., man ABCC11 gene is the determinant for the human earwax type 2013). (Yoshiura et al., 2006) and that human ABCC11 is essential for the secretion of odorants and their precursors from apocrine sweat 2.6. The ABCG family glands (Martin et al., 2009). Finally, human ABCC4 and ABCC7/CFTR clustered with a large clade of arthropod ABCC proteins. Within this The ABCG family is present in most metazoan species, plants clade, 14 T. urticae ABCC transporters formed a sister-group of and fungi but while in metazoans only ABCG HTs have been re- Dappudraft_347288 and a large insect-specific clade containing ported to date, several ABCG FTs (also termed pleiotropic drug lineage-specific expansions of D. melanogaster and T. castaneum resistance proteins (PDRs)) have been identified in plant and fungi ABCC transporters. The function of members from this large (Kovalchuk and Driessen, 2010; Verrier et al., 2008). ABCG HTs have arthropod ABCC transporter clade is mainly unknown. T. urticae a typical reverse domain organization, with the NBD being local- ABCC proteins tetur14g02290, tetur14g02310, tetur14g02320, ized to the N-terminal side of the TMD, and need to dimerize to tetur14g02330 and tetur23g02452 were reported to be down- form a functional transporter. The principal substrates of four out of regulated in diapausing T. urticae females. Diapausing spider mites five human ABCG proteins are endogenous and mostly dietary W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 97

A B

Fig. 5. Phylogenetic analysis of ABCD, E and F proteins of H. sapiens and seven arthropod species. A) ABCD proteins, B) ABCE and F proteins. For procedure and details of phylogenetic analysis see Supplementary Text. For color and shape codes see Fig. 3. The scale bar represents 0.5 amino-acid substitutions per site. lipids, while human ABCG2 (breast cancer resistance protein, BCRP) products of either D. melanogaster scarlet or brown to give func- has a much broader substrate specificity and is known as a multi- tional transporters that are involved in the uptake of pigment drug efflux pump (Kerr et al., 2011; Tarr et al., 2009). In our precursors in cells of Malpighian tubules and developing com- phylogenetic analysis we identified for each arthropod species an pound eyes. Mutation in genes encoding these D. melanogaster ortholog of human ABCG5 and 8. Similar to human ABCG5/ABCG8, ABCG proteins results in flies with eye-color phenotypes (white, the arthropod orthologues (Fig. 6, Table 2), are all found juxtaposed scarlet and brown) distinct from the wild-type brownered-eye- in a head-to-head orientation (Dermauw et al., 2013a). In humans, color phenotype (Ewart et al., 1994; Ewart and Howells, 1998; ABCG5 and 8 are glycoproteins that form obligate heterodimers. Mackenzie et al., 1999). D. melanogaster white mutants with a They limit the absorption of plant sterols and cholesterol from the white-eye color, were the first genetic Drosophila mutants to be diet and promote secretion of plant sterols and cholesterol from isolated and marked the beginning of the Drosophila genetics era liver cells into the bile (Graf et al., 2003; Kerr et al., 2011; Tarr et al., (Green, 1996; Morgan, 1910), but the ABC-transporter function has 2009). Based on their head-to-head orientation and clear ortholo- been attributed to the D. melanogaster white protein only 25 years gous relationships with human ABCG5 and ABCG8, these arthropod ago (Mount, 1987). The role of the T. castaneum and B. mori ortholog ABCGs probably have a similar role as their human orthologues. of D. melanogaster white in eye pigmentation has also been Phylogenetic analysis reveals that D. melanogaster white experimentally validated (Broehan et al., 2013; Komoto et al., clusters with T. castaneum TcABCG-9B, B. mori BGIBMGA002922, 2009). In contrast to insects, both D. pulex and T. urticae have A. mellifera AmABCG2, A. gambiae AGAP000553, and nine ABCG nine co-orthologues of D. melanogaster white. Dimerization be- proteins of both T. urticae and D. pulex. D. melanogaster scarlet has tween these co-orthologues in T. urticae might result in trans- two orthologues in A. gambiae and one in T. castaneum, B. mori, location of pigment precursors to the eyes, while in D. pulex A. mellifera and D. pulex while D. melanogaster brown clusters with dimerization between these co-orthologues or between these co- A. gambiae AGAP007655, A. mellifera AmABCG11 and AmABCG12, orthologues and the D. pulex ortholog of scarlet, might result in and B. mori BGIBMGA002581 and BGIBMGA002712 (Fig. 6, Table 2). transport of pigment precursors to the eyes respectively. However, The D. melanogaster ABCG genes white, scarlet and brown are at as many other functions have been attributed to D. melanogaster present the best studied arthropod ABCG genes. The white and its insect orthologues (Borycz et al., 2008; Zhang and D. melanogaster white gene product heterodimerises with gene Odenwald, 1995; Evans et al., 2008; Komoto et al., 2009), these 98 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110

T. urticae expanded ABCG clade

1:1

D. pulex expanded ABCG clade

Fig. 6. Phylogenetic analysis of ABCG proteins of H. sapiens and seven arthropod species. For procedure and details of phylogenetic analysis see Supplementary Text. For color and shape codes see Fig. 3. The scale bar represents 0.5 amino-acid substitutions per site. One-to-one ABCG orthologues found in H. sapiens and seven arthropod species are shaded in grey, while examples of species-speciﬁc ABCG expansions are indicated with an arch.

proteins might also have alternative functions. Noteworthy, the similar function has been attributed to the T. castaneum ortholog of T. castaneum ortholog of D. melanogaster brown, involved in D. melanogaster E23 (TcABCG-8A) by Broehan et al. (2013). Bryon et transport of red pigment (pteridine) precursors, could not be al. (2013) also reported that tetur17g02510 was upregulated in identified in our phylogenetic analysis. The absence of a clear diapausing T. urticae females compared to non-diapausing females. D. melanogaster brown ortholog in T. castaneum has also been As T. urticae most likely uses ponasterone A instead of 20E as a indirectly demonstrated by Broehan et al. (2013), since injection of molting hormone (Grbic et al., 2011), future research should point TcACBG-9A dsRNA (T. castaneum ortholog of D. melanogaster scarlet out if the T. urticae ortholog of E23 has a similar function as its which is involved in transport of brown pigment (ommochrome) arthropod counterparts. precursors) produces a white-eye instead of a red-eye phenotype. Liu et al. (2011) suggested that BGIBMGA010557 is a B. mori The latter phenotype is in line with Lorenzen et al. (2002) which ortholog of D. melanogaster E23. This orthologous relationship, suggested that the Tribolium larval eyespot and adult eye are only however, could not be deduced from our phylogenetic analysis but pigmented by ommochromes, whose precursors in D. melanogaster instead B. mori BGIBMGA010557 clusters with a group of poorly are normally transported by heterodimers of white and scarlet, and characterized insect ABCG proteins. On the other hand, Liu et al. not pteridines, whose precursors in D. melanogaster are transported (2011) showed that B. mori BGIBMGA010557 (together with by heterodimers of white and brown. BGIBMGA005226, BGIBMGA005203, BGIBMGA005202 and A clear ortholog of D. melanogaster CG3327, also named E23 BGIBMGA010555) is expressed in the midgut and is upregulated by (Early gene at 23), was identified in A. mellifera (AmABCG1), 20E during molting and pupation. For these five B. mori ABCG T. castaneum (TcABCG-8A), D. pulex (Dappudraft1_347416) and proteins we could identify a clear ortholog for almost all insect T. urticae (tetur17g02510) (Fig. 6, Table 2). In D. melanogaster E23 is species included in our analysis (Fig. 6, Table 2). This might suggest a 20-OH ecdysone (20E) induced ABC transporter, probably capable that in insects more than one ABCG protein can be induced by 20E of regulating the 20E response (Hock et al., 2000). Recently, a and consequently that several ABCG proteins might mediate or act W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 99 downstream of 20E responses. The role of these insect ABCG pro- upregulated ABC transporter in two pesticide resistant strains (see teins in insect development has also been shown by Broehan et al. Section 4.2). (2013). Injection of TcABCG-4C (T. castaneum ortholog of B. mori BGIBMGA005202) dsRNA in T. castaneum larvae resulted in arrested 3. Inferring the involvement of ABC transporters in development and rapid death. In addition to this phenotype, xenobiotic transport: methods and assays TcABCG-4C dsRNA injected females laid fewer eggs that failed to hatch. Cryosections of TcABCG-4C dsRNA injected larvae also Human ABC transporters such as ABCB1 (P-gp, MDR1), ABCC2 showed that lipids were depleted in both the larval fat body as the (MRP2) and ABCG2 (BCRP) are extremely well studied for their larval epicuticle. Based on these findings, Broehan et al. (2013) importance in the absorption, distribution and excretion of drugs and suggested that TcABCG-4C is involved in transporting lipids to the therapeutic agents, and consequently a number of tools has been cuticle and is essential for the formation of a waterproof barrier in devised and critically reviewed to study ABC transporters in vivo and the epicuticle. in vitro (Glavinas et al., 2008; Lin and Yamazaki, 2003; Zhang et al., Except for T. urticae, we also identified a clear ortholog of human 2003). Many of the described assays and techniques are directly ABCG1 and ABCG4 in all arthropods included in our analysis (Fig. 6, applicable in the study of arthropod ABC transporters, in particular Table 2). The function of human ABCG4 is not well-known while the in vitro assays using cells and cellular membrane preparations. human ABCG1 has a critical role in controlling intracellular sterol homeostasis in a variety of cells (Kerr et al., 2011; Tarr et al., 2009). 3.1. Whole cell based assays Arthropod orthologues of human ABCG1 have been poorly characterized, with D. melanogaster Atet reported to be expressed in the Whole cell based assays essentially measure the efflux or trachea (Kuwana et al., 1996). Finally, we also identified a lineage- accumulation of compounds in intact cells. Transporters can either specific expansion of ABCG proteins in both D. pulex and T. urticae, be naturally present, for example when investigating drug resis- but found no arthropod orthologues of human ABCG2. No infor- tance cell lines after selection, or can be functionally expressed to mation is available on the function of these expanded groups in high levels in a variety of cells lines by transfection and trans- D. pulex and T. urticae, although in T. urticae some of these formation methods. The active transport of compounds (substrates) responded to xenobiotic exposure (Dermauw et al., 2013a). Human mediated by ABC transporters out of cells results in detectable ABCG2 is known to transport a whole gamut of substrates, lower rate of substrates accumulation, a lower concentration at the comprising anticancer drugs (Kerr et al., 2011; Tarr et al., 2009). steady state equilibrium or a faster rate of substrate elimination in Because of this feature of the human ABCG2 proteins, arthropod pre-loaded cells. Many variations in assay type and a multitude of ABCGs have also been put forward as potential candidates in methods to detect and quantify substrate depletion or accumula- xenobiotic resistance (Buss and Callaghan, 2008; Labbé et al., 2011) tion have been described. Substrates are often labeled (by a fluo- (see Section 4.2). rochrome or radiolabel), but direct and high throughput LC-MS methods have also been devised successfully. This type of assays is 2.7. The ABCH subfamily often used in an indirect set-up. For this purpose, well-known and validated fluorescent dye substrates (such as rhodamine 123, Cal- The ABCH subfamily was first identified in D. melanogaster and is cein AM, Hoechst 33342, tetramethylrosamine (TMR)) are used in not present in mammals, plants and fungi (Dean et al., 2001; competition with candidate substrates (Lebedeva et al., 2011). Cells Kovalchuk and Driessen, 2010; Verrier et al., 2008). Next to ar- expressing ABC proteins are incubated or pre-loaded with these thropods, the zebrafish Danio rerio has also been reported to tractable substrates and eliminated these dyes faster compared to contain an ABCH transporter (Popovic et al., 2010) while other cells lacking ABC-transporter expression. Candidate substrates that teleost fish lack this type of ABC transporter (Liu et al., 2013). In our are transported are identified by their potential to interfere with analysis clear orthologous relationships were detected between all dye transport kinetics. In a similar set-up, inhibitors of ABC trans- insect ABCH transporters, with each insect species having three porters can be identified. ABCH transporters (Table 2, see Supplementary Fig. 2). The Cellular assays have also been devised using cell mono-layers,as diamond-back moth Plutella xylostella has been reported to contain this is of particular importance when in vivo pharmacological bar- twoefour copies (depending on the presence or absence of allelic riers such as intestinal epithelial cells are taken into account in drug variation in the dataset) of the D. melanogaster ABCH protein transport. However, in such experiments cell lines forming a CG9990 (You et al., 2013). In contrast to insects, D. pulex and confluent monolayer with very tight junctions are needed, which T. urticae have a relatively high number of ABCH proteins (15 and complicates these experiments (Proctor et al., 2010). 22, respectively). Both D. pulex and T. urticae ABCHs clustered into separate clades (Table 2, see Supplementary Fig. 2), suggesting that 3.2. Vesicular assays the diversity of ABCH proteins in these species has been due to lineage-specific expansions ((Dermauw et al., 2013a), this study). Vesicular transport assays are membrane based assays that are Similar to ABCG proteins, ABCH proteins are HTs and have a reverse often used to detect and quantify the translocation of molecules by domain organization, with the NBD at the N-terminal side of the ABC transporters. The power of the assay is based on the revolu- TMD. Their function, however, is poorly understood. tionizing work of Steck and Kant (1972), who describe the prepa- D. melanogaster CG33970 is 2-fold upregulated after cold hardening ration of ‘inside out vesicles’, which can be separated from other of adult flies (Qin et al., 2005) while six ABCH genes were differ- membrane fragment such as open membrane lamellae and ‘right entially expressed in diapausing T. urticae females compared to side out’ vesicles by density gradient centrifugation. In these vesi- non-diapausing females (Bryon et al., 2013). D. melanogaster cles, the ATP-binding site and substrate binding site of the ABC CG9990 null mutants and an RNAi line that silences D. melanogaster transporters are facing outwards (to the assay buffer solution). CG9990 are both lethal (Mummery-Widmer et al., 2009; Zhang Consequently, the transport of substrates into vesicles can be et al., 2009) while TcABCH-9C dsRNA injection in T. castaneum studied, which is essentially more straightforward compared to larvae resulted in similar phenotypes as injection with TcABCG-4C quantifying substrate depletion or deviations from steady state (see Section 2.6). Of peculiar interest, a P. xylostella ortholog of equilibrium in whole cell based assays. After substrate incubation, D. melanogaster CG9990 ((You et al., 2013), this study) was the most vesicle preparations are isolated from the assay solution by rapid 100 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110

filtration using glass fiber filters or nitrocellulose membranes and 3.6. In vivo assays the amount of substrates inside vesicles can be quantified by similar methods as described above (Krumpochova et al., 2012). Next to the in vitro assays described above, a number of in vivo This assay has been successfully performed with a number of tools has also been used to link ABC-transporter activity to a membrane preparations from different sources, including insect certain drug resistant phenotype, such as the generation of cells. Similar to whole cell based assays, vesicular membrane assays (double) knock-out and mutant mice and rat strains (reviewed in can also be used in an indirect set-up where test compounds Lin and Yamazaki, 2003; Zhang et al., 2003). However, given the interfere with the uptake of known and easy to track model sub- lack of orthologues relationships between most ABC proteins strate molecules (Glavinas et al., 2008; Krumpochova et al., 2012; across species (and especially between vertebrates and in- Lin and Yamazaki, 2003; Zhang et al., 2003). vertebrates) (Table 2), such models are of limited use as a technique to assign function to arthropod ABC proteins. Unfortunately, 3.3. ATPase assays this is also true for most ABC transporters between different insect species (Table 2) and limits the use of D. melanogaster as an The ATPase assay is another type of in vitro assay that is used arthropod genetic model, except for the rare cases where clear fi frequently. It is based on the link between ATP hydrolysis and sub- orthologues can be identi ed. Proof of principle that gene knock- strate transport. In these assays, the hydrolysis of ATP during sub- out by P-element insertion or by constructing RNAi lines can be strate transport is followed by quantification of the liberated used to study ABC-transporter function in Drosophila has been inorganic pyrophosphate. Compounds that are transported (or recently delivered (Chahine et al., 2012). More generally, reverse interfere with transport, such as inhibitors) modulate this ATPase genetics by RNAi (either by injection or oral administration) can activity (Litman et al., 1997). The rather low sensitivity of the assay be a powerful tool in assessing ABC function in non-classical requires high levels of ABC proteins and thus inside out vesicle models, as was recently very elegantly demonstrated for preparations, prepared from transfected or transformed cell lines, T. castaneum (Broehan et al., 2013). are most frequently used. In this set-up, also open lamellae in ve- Last, chemosensitizers that inhibit ABC transporters have been sicular preparations contribute to the measured activity. ATPase discovered a long time ago and have found clinical applications in activity is vanadate sensitive, and a fast transport of substrates re- overcoming multidrug resistance in humans (reviewed in Choi, sults in a reproducible and strong increase of ATPase activity. Sub- 2005), but they are also potentially useful experimental tools in strates that are transported slowly, do not generally cause a linking a resistant phenotype to the action of an ABC trans- detectable increase in ATPase activity, but can be identified when porters. Chemosensitizers or synergists are administered used as competitive substrates in an assay with fast transported together with a toxicant or drug, and restore the toxicity or ef- fi fi model substrates (Glavinas et al., 2008; Lin and Yamazaki, 2003; cacy both in vivo and in vitro. One of the rst compounds 2þ þ Zhang et al., 2003). These cell and membrane assays are conceptu- discovered was verapamil, a phenylalkylamine Ca and K fi ally simple and interpretation of results is usually straightforward. channel blocker with wide clinical use. Verapamil was rst shown to reverse multidrug resistance in neoplastic cells and chloroquine resistant Plasmodium falciparum (Martin et al., 1987). 3.4. Nucleotide trapping assay It has since then been widely used in the study of mainly human P-gp where it acts as a competitive substrate inhibitor, but it is The nucleotide trapping assay is based on the occurrence of a less potent in inhibiting human MRP1, although it stimulates transition state complex during transport that contains an MRP1 mediated glutathione transport (Loe et al., 2000). Although occluded nucleotide in the nucleotide-binding site (Urbatsch verapamil has been used as a synergist in insecticide resistance, et al., 1995). This state can be stabilized by the addition of the efficiency and specificity of verapamil as a competing sub- vanadate. The amount of nucleotides trapped is proportional to strate or inhibitor in distantly related arthropod ABC transporters fi the rate of transport, and can be quanti ed by incubations with is still largely unknown. 32P labeled azido ATP. This method is especially useful for ABC transporters where ATPase activity is not stimulated by substrate 3.7. Genetic mapping addition and where vesicular assays cannot be used (for example fl for the so called ABC ippases, like human ABCB4) (Smith et al., The association between ABC transporters and xenobiotic 2000). resistance is often based on synergism of toxicity, combined with the observation of increased expression (see Table 3). However, 3.5. Fluorescence spectroscopic tools single point mutations in a transmembrane domain can modulate transport and selectivity of ABC transporters. A SerePhe substitu- A number of fluorescence spectroscopic tools has also been tion for example drastically altered the overall degree of drug described to study ABC transporters and to detect potential sub- resistance and specificity conveyed by mdr1 and mdr3 in mice strates. Transporters can be labeled with sulfhydryl-specific fluo- (Gros et al., 1991). If synergism points out the role of ABC trans- rescent probes on the two conserved Cys residues in the Walker A porters in resistance, such point mutations cannot be detected by motif of the nucleotide-binding domain. The fluorescent groups are expression analysis and unbiased approaches are needed. The covalently linked and upon nucleotide binding (ATP, ADP and an- availability of genetic mapping tools in Drosophila and some lepi- alogs) a saturable quenching of fluorescence can be detected, dopteran species including B. mori, have proven powerful in finding indicative of transport (Sharom et al., 2001; Sreeramulu et al., resistance loci associated with ABC transporters (Begun and 2007). Another technique is based on the conservation of trypto- Whitley, 2000; Heckel, 2012) (see also Section 4.4.2). Until phan (Trp) residues in proteins across the ABC family. Within pu- recently, such approaches were limited to arthropods where clas- rified ABC transporters, those Trp residues contribute to sical genetic resources are available, such as genetic linkage maps. fluorescence which is quenched upon binding of trinitrophenyl However, it was recently shown how classical bulk segregant nucleotides. Trp fluorescence is also very sensitive to binding of analysis can be modified to allow ultra high resolution mapping in substrates, inhibitors and other modulators (Liu et al., 2000; any arthropods species for which classical genetic resources are Sharom et al., 2001). sparse, but a genome sequence is available ((Van Leeuwen et al., Table 3 An overview of reported associations between ABC transporters and insecticide/acaricide transport and resistance.

Pesticide class Pesticide Species ABC subfamily E/syn a E/exp E/oth Evidence Reference Carbamates Pirimicarb M. persicae ABCG/ABCHb v Increased expression (microarray) in adult aphids upon Silva et al., 2012 exposure to pirimicarb Thiodicarb H. virescens ABCB FT (P-gp) v v Quinidine synergism (synergism ratio of 12.5 and 1.8 in Lanning et al., 1996a,b resistant and susceptible strain, respectively); increased P-gp expression in thiodicarb resistant larvae (Western Blot) Macrocyclic Abamectin R. microplus ? v Cyclosporin A and MK571 synergism Pohl et al., 2012 lactones Abamectin D. melanogaster ABCB FT (P-gp) v v v Increased P-gp expression (Western Blot) and increased P-gp Luo et al., 2013a related vanadate-sensitive ATPase activity in abamectin resistant strain; verapamil synergism; higher P-gp expression (Western Blot, C219 antibodies) in the humoral/CNS interface in

abamectin resistant strain 89 (2014) 45 Biology Molecular and Biochemistry Insect / Leeuwen Van T. Dermauw, W. Avermectin D. melanogaster ABCB FT (P-gp) v v Induction of P-gP expression (Western Blot) and increased P-gp Luo et al., 2013b related ATPase activity upon exposure of S2 cells to avermectin; avermectin enhances cellular efﬂux of rhodamine 123 from S2 cells Emamectin L. salmonis ABCB FT (P-gp)/? v v Verapamil synergism of emamectin benzoate (EMB); induction Igboeli et al., 2012; Tribble benzoate of P-gp expression (qPCR) upon exposure to EMB et al., 2007 Ivermectin P. humanus ABCB_unknown/ v v v Increased expression (qPCR) of six ABC transporters in female Yoon et al., 2011 humanus ABCC/ABCG lice 4 h post-ivermectin exposure by both immersion and contact applications; injection of PhABCC4 dsRNA (P. humanus ortholog of CG6214) resulted in increased sensitivity to ivermectin; verapamil synergism C. pipiens ? v Verapamil synergism in 4th instar larvae Buss et al., 2002 R. microplus ABCB HT v v Cyclosporin A and MK571 synergism, increased expression Pohl et al., 2011, 2012 (qPCR) of RmABCB10 in ivermectin (IVM) resistant females and RmABCB10 transcription was upregulated in IVM-resistant females in response to IVM feeding S. scabiei ABCC v Increased expression (qPCR) upon exposure to ivermectin Mounsey et al., 2007 C. riparius ? v v Verapamil and cyclosporin synergism; ivermectin stimulates Podsiadlowski et al., 1998 ATPase activity in homogenate fractions of C. riparius larvae Moxidectin R. microplus ? v Cyclosporin A and MK571 synergism Pohl et al., 2012 Neonicotinoids Acetamiprid A. mellifera ? v Verapamil synergism Hawthorne and Dively 2011 Imidacloprid A. mellifera ? v Verapamil synergism Hawthorne and Dively 2011 Thiacloprid A. mellifera ? v Verapamil synergism Hawthorne and Dively 2011 Thiametoxam B. tabaci ABCG v Increased expression (microarray) in thiamethoxam resistant Yang et al., 2013a,b

strain e Strycharz 2010, Strycharz 110 Organochlorines DDT A. arabiensis ABCB FT (P-gp)/ABCG v Increased expression (qPCR) in a DDT-resistant strain Jones et al., 2012 et al., 2013 D. melanogaster ABCB FT (P-gp)/ABCC v v v Increased expression (qPCR) in a DDT-resistant strain (91-R), verapamil synergism (synergism ratio of 10 and 1.1 in resistant RNAi results in 91-R ﬂies being more susceptible to DDT, and susceptible strain, respectively) D. melanogaster ABCG v Increased expression (microarray) in DDT-resistant strains (91- Pedra et al., 2004 R and Wisconsin) Endosulfan C. pipiens ? v Verapamil synergism in 4th instar larvae Buss et al., 2002 H armigera ABCB FT (P-gp) v Stimulation of ATPase activity; TMR transport declined with Srinivas et al., 2005; Aurade increasing concentrations of insecticide; Trp quenching et al., 2006, 2010b Organophosphates Chlorpyrifos R. microplus ? v Cyclosporin A and MK571 synergism Pohl et al., 2012 P. xylostella ABCA/ABCC/ABCG/ v Increased expression in chlorpyrifos resistant strain (RNA-seq) You et al., 2013 ABCH/ABCF Coumaphos A. mellifera ? v Verapamil and oxytetracycline synergism Hawthorne and Dively 2011

(continued on next page) 101 Table 3 (continued ) 102

Pesticide class Pesticide Species ABC subfamily E/syn a E/exp E/oth Evidence Reference Ethylparaoxon H. armigera ABCB FT (P-gp) v Stimulation of ATPase activity; TMR transport declined with Aurade et al., 2010b increasing concentrations of insecticide; Trp quenching Methylparathion H. armigera ABCB FT (P-gp) v Stimulation of ATPase activity; TMR transport declined with Srinivas et al., 2005; Aurade increasing concentrations of insecticide; Trp quenching et al., 2006, 2010b Monocrotophos H. armigera ABCB FT (P-gp) v Stimulation of ATPase activity; TMR transport declined with Srinivas et al., 2005; Aurade increasing concentrations of insecticide; Trp quenching et al., 2006, 2010b Temephos A. caspius ? v Verapamil synergism (by factor 3.5) Porretta et al., 2008 A. aegypti ABCB FT (P-gp) v v v Increased expression (qPCR) in larvae upon exposure to Figueira-Mansur et al., 2013 temephos; RNAi of P-gp resulted in a signiﬁcant increase in temephos toxicity to larvae, verapamil synergism Triazophos N. lugens ABCC v Increased expression (SSH and qPCR) in nymphs exposed to Bao et al., 2010

sublethal concentrations of triazophos 89 (2014) 45 Biology Molecular and Biochemistry Insect / Leeuwen Van T. Dermauw, W. Pyrethroids Cyfluthrin C. lectularius ? v RNAi resulted in reduced resistance to beta-cyfluthrin in a Zhu et al., 2013 deltamethrin resistant strain Cypermethrin C. pipiens ? v Verapamil synergism in 4th instar larva Buss et al., 2002 H. armigera ABCB FT (P-gp) v Stimulation of ATPase activity; TMR transport declined with Srinivas et al., 2005; Aurade increasing concentrations of insecticide; Trp quenching et al., 2006, 2010b Deltamethrin C. lectularius ? v Increased expression of eight ABC transporters (RNA-seq and Mamidala et al., 2012 qPCR) in a deltamethrin resistant strain; an ABCG transporter was the highest upregulated ABC-transporter A. gambiae ABCC v Increased expression (RNA-seq) in deltamethrin resistant strain Bonizzoni et al., 2012 T. ni ABCB FT (P-gp) v Limited increased expression (qPCR) in insects fed on a Simmons et al., 2013 deltamethrin containing artificial diet C. lectularius ? v Increased expression of four ABC transporters in a deltamethrin Zhu et al., 2013 resistant strain (RNA-seq and qPCR) Fenvalerate H. armigera ? v Stimulation of ATPase activity Srinivas et al., 2005, Aurade et al., 2006 Fluvalinate A. mellifera ? v Verapamil and oxytetracycline synergism Hawthorne and Dively 2011 Pyrethroids A. aegypti ABCB HT/ABCC/ABCG v Increased expression (microarray and qPCR) in pyrethroid Bariami et al., 2012 resistant strains; ABCB4 gene has a higher copy number in pyrethroid resistant strains b Diverse Diflubenzuron A. caspius ? vv v VerapamilA deletion or synergism insertion (by in the factor ABCC2 16.4) gene is correlated with both PorrettaHeckel 2012, et al., Lei 2008 et al., 2013 Cry1A toxin Lepidoptera ABCA/ABCB/ABCC/ABCD/ABCG higher Bt resistance levels and loss Cry1A binding to midgut membranes; eight ABC genes were differentially expressed between Cry1Ac-resistant and -susceptible larvae of P. xylostella Fipronil P. xylostella ABCA/ABCC/ABCG/ABCH v Increased expression (RNA-seq) in fipronil resistant strain You et al., 2013 Multiresistant T. urticae ABCC/ABCH v Increased expression (microarray) in two multiresistant strains Dermauw et al., 2013a,b Multiresistant H. armigera ABCB FT (P-gp) v Increased P-gp expression (Western Blot) in multiresistant Srinivas et al., 2004

larvae e 110 a Type of evidence was divided into three categories: E/syn, ABC inhibitor synergism; E/exp, increased expression of ABC genes; E/oth, other type of evidence. b ABC subfamily was determined in this study and based on the top BLASTp hits in the NCBI non redundant protein database. W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 103

2012), see also Section 4.4.1). This technique relies on next gener- transport assays that directly quantify insecticide transport have ation re-sequencing of selected populations and seems especially not been reported until present, although many of the methods timely with thousands of arthropods genomes being sequenced using Sf9 cells to recombinantly express ABC transporters and currently (i5K-Consortium, 2013). prepare vesicles are directly relevant for arthropod studies (see Section 3). 4. Arthropod ABC transporters in xenobiotic transport and resistance 4.2.1. Carbamates and organophosphates Lanning et al. (1996a) showed for the first time a link between 4.1. ABC transporters as players in detoxification pathways an arthropod ABC transporter and resistance against an insecticide, the carbamate thiodicarb (Table 3). Using human monoclonal Arthropods have developed a variety of mechanisms to cope P-gp antibodies, it was demonstrated that ABCB FTs/P-gps were with toxic compounds. These mechanisms are generally classified highly expressed in the cuticle of larvae of the tobacco budworm in mechanisms of decreased response to toxic substances, often Heliothis virescens and that P-gps were overexpressed in thio- referred to as pharmacodynamic mechanisms (interaction of toxic dicarb resistant larvae of this species. Furthermore it was found substances with their target site) and mechanisms of decreased that inhibition of ABCB FTs/P-gps with quinidine decreased the exposure also called pharmacokinetic mechanisms, which include LD50 of thiodicarb for resistant H. virescens larvae by a factor of altered penetration, distribution, metabolism and excretion of the 12.5, while that of susceptible larvae only decreased by a factor of toxic substances. In most cases, high levels of toxicant resistance 1.8. In a follow-up paper, it was further shown that thiodicarb in arthropods is due to alterations in the sensitivity of the target treatment in combination with quinidine increased C14 labeled site caused by point mutations (pharmacodynamic mechanism), thiodicarb accumulation 2-e3-fold as compared to thiodicarb or the efficient metabolism or sequestration of the toxic com- treatment alone (Lanning et al., 1996b). Other reports have asso- pound (pharmacokinetic mechanism). The overall detoxification ciated ABC transporters with carbamate resistance, but without process can be divided into three phases, phase IeIII. In Phase I further characterization. Genes encoding an ABCG and ABCH reactions, enzymes such as P450 monooxygenases and carbox- transporter were among the 183 upregulated genes in adults of ylesterases activate the toxic molecules and make them more the green peach aphid Myzus persicae exposed to pirimicarb. The reactive and water soluble by introducing hydroxyl, carboxyl and upregulation of these ABC transporters was however not validated amino groups, which often results in an increased conjugation by qRT-PCR and no synergism tests were performed (Silva et al., with endogenous molecules by phase II enzymes such as GSTs or 2012). Several studies have also reported on the ABCB FT/P-gp- UPD-glycosyltransferases. Finally, in phase III the polar com- mediated efflux of organophosphates (OPs) (Aurade et al., 2006, pounds or conjugates can be transported out of the cell by cellular 2010b; Srinivas et al., 2005). Aurade et al. (2006, 2010b) found transporters and it is in this phase that ABC transporters play an that ethylparaoxon, monocrotophos and methylparathion at a important role. In some cases cellular transporters such as ABCs concentration of 100, 100 and 50 mM stimulated purified P-gp- directly and efficiently transport toxicants out of the cell without mediated ATPase activity by 20%, 25% and 30% in the tobacco enzymatic modifications, thereby preventing the exertion of budworm Helicoverpa armigera, respectively, suggestive of P-gp toxicity in the cell or organism (sometimes also referred to as meditated transfer of these compounds (see Section 3). In this “Phase 0”)(Despres et al., 2007; Kennedy and Tierney, 2013). ABC study, it was also shown by Trp fluorescence quenching that transporters have been implicated in resistant phenotypes in ethylparaoxon and methylparathion interact with H. armigera P- many animals, including both vertebrates and invertebrates, and gp with relatively high affinity (Kd values of 25 and 50 mM). Finally, are in the latter group probably best studied in nematodes the authors also succeeded in reconstituting H. armigera P-gp into (Ardelli, 2013; Lespine et al., 2012). Fewer cases have been re- proteoliposomes and showed that ethylparaoxon, monocrotophos ported in arthropod resistance, although the interest in this family and methylparathion inhibit P-gp-mediated transport of the is steadily growing, especially with the availability of more fluorescent reporter dye tetramethylrosamine (TMR) in a con- sequence data and the availability of genetic tools in organisms centration dependent manner. Whether OPs compete with TMR other than Drosophila. A survey of cases where the involvement of as substrates or act as non-competitive inhibitors was not ABC transporters in arthropod resistance is suggested is presented demonstrated. Interestingly, in a follow-up study it was shown in Table 3 and discussed below in the light of the strength of the that cholesterol could increase the ethylparaoxon or methylpar- evidence. athion stimulated H. armigera P-gp ATPase activity in recon- stituted proteoliposomes by 10e20% (Aurade et al., 2012). Another 4.2. ABC transporters associated with insecticide transport and example of interaction between ABCB FTs/P-gps and OPs was resistance recently documented for the mosquito Aedes aegypti (Figueira- Mansur et al., 2013). An 8-fold increase of an A. aegypti ABCB ABC transporters have been associated with transport of and/or FT/P-gp gene was observed in A. aegypti larvae 48 h after treat- resistance to 27 different insecticides/acaricides, belonging to nine ment with 0.0007 mg/L temephos. Furthermore, temephos distinct chemical classes and several mode of action groups: car- treatment of A. aegypti larvae in combination with a sublethal bamates, macrocyclic lactones, neonicotinoids, organophosphates, dose of verapamil resulted in a decrease of the LC50 of temephos pyrethroids, cyclodienes, benzoylureas, phenylpyrazoles, and DDT by a factor of 1.24 and temephos toxicity increased by 57% in P-gp (reviewed in (Buss and Callaghan, 2008) and updated in this study, silenced larvae. Similarly, Porretta et al. (2008) showed that P-gp Table 3). ABCB FTs/P-gps followed by ABCCs and ABCGs were the inhibition by verapamil in the closely related Aedes caspius most reported ABC subfamilies to be involved in pesticide transport resulted in LD50 reduction by a factor of 3.5. These results suggest and/or resistance. The evidence pointing toward a role in transport a role for P-gp in efflux of temephos in Aedes sp., but whether or resistance is most often based on the quantification of transcript these P-gps are also involved in temephos resistance in these or protein levels, and by synergism studies using ABC inhibitors species remains to be determined. Finally, a link between ABC that are poorly characterized in arthropods. In some cases, ATPase transporters in OP efflux has also been suggested for two other assays revealed the simulation of ATP hydrolysis upon contact with arthropod species. In a chlorpyrifos resistant strain of P. xylostella insecticides, which is considered indicative of transport. Vesicular ABCA/C/G/H and F transporter(s) were more highly expressed 104 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 compared to a susceptible strain as determined by RNA-seq (You Furthermore, exposure of tick females to ivermectin resulted in an et al., 2013) while in the brown planthopper Nilaparvata lugens upregulation of RmABCC10 in the resistant strain while transcript SSH and qRT-PCR revealed a significant increase of an ABCC levels did not significantly change in females of the susceptible transporter in nymphs exposed to sublethal concentrations of strain (Pohl et al., 2011, 2012). ABCB HTs are located in the mito- triazophos (Bao et al., 2010). However, whether increased chondria and are believed to play a role in iron homeostasis and expression is related to transport and resistance has not been protection against oxidative stress. However, contrary to ABCB FTs demonstrated. they have rarely been linked with xenobiotic resistance (Elliott and Ai-Hajj, 2009; Zutz et al., 2009). 4.2.2. Macrocyclic lactones The most convincing evidence for the involvement of ABC Resistance to macrocyclic lactones (MLs), such as abamectins, transporters in ML resistance has been published recently by Luo ivermectin and moxidectins, in arthropods and nematodes is in et al. (2013a,b). It was shown that avermectin stimulated P-gp most cases linked to several genetic factors. Both mutations in ATPase activity and efflux of Rhodamine 123 in Drosophila derived target-site genes (e.g. in glutamate-gate chloride channels), as well S2 cells and that ABCB FTs/P-gp expression was induced by aver- as quantitative changes in the expression of detoxification enzymes mectin in a dose- and time-dependent manner. Next, it was shown and ABC transporters have been reported to be involved in ML that avermectin induced P-gp expression was caused by an eleva- resistance (Dermauw et al., 2012; Pu et al., 2010; Wolstenholme and tion of intracellular calcium levels, both directly by activating cal- Kaplan, 2012)(Table 3). In mammals, it has been clearly demon- cium ion channels, and indirectly by activating chloride channels. strated that ivermectin is a substrate of P-gps as human MDR-cells Of particular interest, a well-known chloride channel agonist accumulate less 3H ivermectin (Pouliot et al., 1997; Schinkel et al., pentobarbital enhanced ivermectin induced P-gp expression in a 1994). The role of ABC transporters in ML transport and/or resis- concentration dependent manner. Induced expression by the tance in nematodes has been thoroughly characterized, with the closely related chemical structure phenobarbital of two ABCB FTs/ majority of MLs stimulating ABCB FT/P-gp-mediated efflux of the P-gps and an ABCC transporter (CG6214) in D. melanogaster flies reported substrate Rhodamine 123 (Kerboeuf and Guégnard, 2011). was recently also demonstrated by Misra et al. (2011). In a follow- In contrast, the involvement of ABCB FT/P-gps in ML transport and/ up study of Luo et al. (2013a,b), the role of ABCB FTs/P-gps in aba- or resistance in arthropods is not well documented. Podsiadlowski mectin resistance in D. melanogaster was investigated using a 7-fold et al. (1998) reported for the first time the synergistic action of abamectin resistant strain that was selected from a susceptible ABCB FT/P-gp-transporter inhibitors on the toxicity of a ML in an laboratory strain (Canton-S). ABCB FT/P-gp expression and P-gp insect species. Sublethal concentrations of verapamil (30 mM) or ATPase activity in the resistant strain was increased 2-e3-fold cyclosporin A (3 mM) reduced the LC50 of ivermectin for the midge compared to the susceptible strain. Treatment with verapamil Chironomius riparius with a factor 2.7 and 2.8, respectively. reduced P-gp ATPase activity in the adults of the resistant strain by Furthermore, it was shown that P-gps were located in the anal 89% compared to 31% in the susceptible strain, while abamectin papillae of C. riparius larvae and that ivermectin at a concentration LD100 values for resistant and susceptible larvae were reduced by of 0.1e1 mM stimulated ABCB FT/P-gp-mediated ATPase activity in 84% and 33% in the same treatment. Through confocal microscopy homogenate fractions of C. riparius larvae. Similar to C. riparius, and immunofluorescent staining with human anti-P-gp C219 an- 100 mM verapamil has also been shown to synergistically enhance tibodies, it was also observed that P-gp expression in the bloode the toxicity of ivermectin in the mosquito Culex pipiens. Comparison brain barrier (BBB) in heads from adults of the resistant strain was of synergism ratios between resistant and susceptible strains did not higher than that of the susceptible strain. Finally, avermectin reveal any differences, indicating that ABC transporters did not treatment resulted in an increased P-gp expression in the BBB of contribute to resistance (Buss et al., 2002). In 2012, Igboeli et al. the resistant strain, compared to a decline in the susceptible strain. found that concurrent exposure of the sea lice Lepeophtheirus sal- Taken all this data into account, it is likely that P-gps are involved in monis (Crustacea: Copepoda) to 10 mM verapamil and 300 mg/L (low-levels) of abamectin resistance in D. melanogaster. emamectin benzoate (EMB) caused significantly higher mortality compared to exposure of L. salmonis to EMB alone. In addition, ABCB 4.2.3. Organochlorines FT/P-gp expression was induced in adult sea lice exposed to 1 mg/L Associations between DDT resistance and ABC transporters are EMB. Induced expression of ABC transporters upon exposure to scarce. An ABCG transporter gene was significantly overtranscribed ivermectin was also observed in females of the body louse in resistant strains of D. melanogaster and Anopheles arabiensis P. humanus and the scabies mite Sarcoptes scabiei (Mounsey et al., (Jones et al., 2012; Pedra et al., 2004). Recently, Strycharz et al. 2007, 2006; Yoon et al., 2011). For the body louse, it was also (2013) determined that D. melanogaster flies from a highly resis- shown that injection of dsRNA of P. humanus ABCC4 (the most highly tant DDT strain (91-R) excreted w4-fold more DDT and its metab- expressed ABC gene after exposure to ivermectin) into female lice olites than flies from the susceptible strain. A topical pretreatment increased their sensitivity to ivermectin with 20e30% compared to with verapamil reduced the LC50 value of the resistant flies by 10- buffer injected lice (Yoon et al., 2011). However, the low number of fold while no effect on the LC50 of susceptible flies was observed. lice injected (n ¼ 15) and the discrepancy between assessing toxicity In a preliminary report, Strycharz et al. also showed that two ABCB (12 h post injection) and the reduction in transcript levels (48 h post (mdr50 and mdr65) and one ABCC (dMRP/CG6214) transporter injection) require further validation. gene(s) were overexpressed (w1.3-fold) in the resistant The role of ABC transporters in ML resistance has at present only D. melanogaster strain compared to the susceptible strain. RNAi been thoroughly investigated in two arthropod species, the cattle knockdown of these genes in resistant flies also increased their tick Rhipicephalus microplus and the fruit fly D. melanogaster (Luo susceptibility to DDT (Strycharz, 2010; Strycharz et al., 2010). Taken et al., 2013a; Pohl et al., 2011, 2012). Pretreatment with 5 mMof together, ABC transporters are most likely involved in the increased ABC-transporter inhibitors cyclosporin A and MK571 reduced the excretion of DDT in D. melanogaster (91-R) resistant flies, hereby LC50 of ivermectin, abamectin and moxidectin in field collected lowering the concentration of DDT reaching the target. The role of resistant R. microplus larvae with a factor of 1.2e1.5, while toxicity ABC transporters in transport/resistance of other organochlorines was not affected in a susceptible laboratory strain. Resistant has only been marginally studied. Verapamil increased toxicity to R. microplus females showed a higher constitutive expression of an endosulfan in 4th instar larvae of the mosquito C. pipiens (Buss ABCB HT gene (RmABCB10) compared to the susceptible strain. et al., 2002) while endosulfan stimulated ATPase activity and W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 105

Table 4 An overview of reported associations between ABC transporters and allelochemical transport.

Allelochemical Species ABC subfamily Evidence Reference

? T. urticae ABCA/ABCC/ABCE/ABCG/ABCH Increased expression (RNA-seq) upon transfer from preferred host Grbic et al., 2011, (bean) to a more challenging host (tomato) Dermauw et al., 2013a ? Dendroctonus ABCGa Increased expression (RNA-seq) in adults that were fed on lodgepole Robert et al., 2013 ponderosae pine host tree tissues compared to adults that were starved Cardenolide M. sexta ? Verapamil inhibits P-gp-mediated efﬂux of digoxin at the perineurium Petschenka et al., 2013 of nerve cords Colchicine D. melanogaster ABCB FT(P-gp) Expression of mdr49 in brain and gut of larvae is enhanced upon Tapadia and Lakhotia colchicine feeding (as determined by Northern blotting); 2005; Wu et al., 1991 Drosophila mdr49 deletion mutants showed increased sensitivity to colchicine Curcumin H. armigera ABCB FT(P-gp) Curcumin mixture inhibited the activity of H. armigera P-gp ATPase by Aurade et al., 2010a 80e90% at 100 mM concentration, addition of the curcuminoid mixture collapsed the tetramethylrosamine concentration gradient generated by H. armigera P-gp ATPase Flavonoids H. armigera ABCB FT(P-gp) Flavonoids (morin, quercetin and phloroglucinol) inhibited H. armigera Aurade et al., 2011 P-gp ATPase activity and bind to H. armigera P-gp with high afﬁnity Nicotine M. sexta ? Verapamil inhibits accumulation of nicotine in the lumen of Malpighian Murray et al., 1994, tubules; ABC transporters were downregulated (microarray) in M. sexta Gaertner et al., 1998, larvae fed on tobacco plants transformed to silence nicotine Govind et al., 2010 biosynthesis compared to larvae that fed on wild-type tobacco plants Vinblastine M. sexta ? Verapamil inhibits accumulation of vinblastine in the lumen of Gaertner et al., 1998 Malpighian tubules

a ABC subfamily was determined in this study and based on the top BLASTp hit in the NCBI non redundant protein database. inhibited transport of tetramethylrosamine in H. armigera (Aurade upregulated in adult Bemisia tabaci flies of a thiamethoxam recent et al., 2006, 2010b; Srinivas et al., 2005). Sreeramulu et al. (2007) strain (Yang et al., 2013a,b). Hawthorne and Dively (2011),onthe observed similar effects of endosulfan on ABCB FTs/P-gps of hu- other hand, showed that verapamil synergized honeybee mortality man multidrug resistant cells. to three neonicotinoids: acetamiprid, imidacloprid and thiacloprid but necessary experiments to link this finding to neonicotinoid 4.2.4. Pyrethroids resistance were not performed. Verapamil has also been shown to fl For three different insect species, pyrethroid resistance has also enhance the toxicity of di ubenzuron, a chitin synthesis inhibitor, been associated with the upregulation of ABC-transporter genes by a factor of 16.4 in 4th instar larvae of the mosquito A. caspius (Bariami et al., 2012; Bonizzoni et al., 2012; Mamidala et al., 2012; (Porretta et al., 2008). Finally, several ABC transporters (belonging fi Zhu et al., 2013). Bariami et al. (2012) showed by microarray ex- to ve different ABC subfamilies) were upregulated in a P. xylostella fi periments that a P450 monooxygenase (Cyp9J26) and an ABCB strain resistant against pronil, an insecticide belonging to the transporter gene (ABCB4), were upregulated in a pyrethroid resis- chemical class of phenylpyrazoles (You et al., 2013). tant strain of the dengue vector A. aegypti. Furthermore, quantitative PCR revealed that increased transcript levels of these A. aegypti 4.3. ABC transporters involved in transport of plant secondary w genes were associated with a 7-fold increase in gene copy compounds (allelochemicals) number. A. aegypti ABCB4 belongs to the ABCB half transporters, which have only very rarely been associated with xenobiotic For several plant pathogenic fungi, it has been shown that ABC resistance but are thought to be mitochondrial ABC transporters transporters mediate cellular tolerance to natural toxic plant e with roles in the biogenesis of cytosolic iron sulfur clusters, heme compounds and/or virulence (Coleman and Mylonakis, 2009; biosynthesis, iron homeostasis and protection against oxidative Gardiner et al., 2013). Compared to fungi however, transport of stress (Elliott and Ai-Hajj, 2009; Zutz et al., 2009). In another plant allelochemicals by arthropod ABC transporters has only been transcriptome study, RNA-seq analysis and qPCR revealed that four marginally studied (Sorensen and Dearing, 2006)(Table 4). About e ABC transporters (ABC8 11) were upregulated in a deltamethrin 15 years ago it was shown that verapamil inhibited transport of resistant strain of Cimex lectularius when compared to a susceptible nicotine and vinblastine to the lumen of Malpighian tubules of the strain. In addition, RNAi mediated knockdown of two of these ABC tobacco hornworm Manduca sexta, indicating the involvement of an genes (ABC8 and 9) resulted in an increased susceptibility to beta- ABCB FT/P-gp transporter in the excretion of alkaloids in this lepi- fl cy uthrin in deltamethrin resistant female bed bugs (Zhu et al., dopteran species. A similar ABC transporter for nicotine excretion 2013). However, in some species there is little to no relationship was also suggested to regulate transport over the BBB of the same between pyrethroid exposure and increased expression of ABCB insect species (Gaertner et al., 1998; Murray et al., 1994). In line FTs/P-gp as recently shown by Simmons et al. (2013) for larvae of with the latter findings, Govind et al. (2010) reported that ABC T. ni. Besides alterations in ABCB FT/P-gp expression levels, pyre- transporters were downregulated in M. sexta larvae that fed on throid stimulation of ATPase activity and verapamil synergism of tobacco plants that were transformed to silence nicotine biosyn- pyrethroid toxicity has been reported for several insect species thesis compared to larvae that fed on wild-type tobacco plants. (Aurade et al., 2006, 2010b; Buss et al., 2002; Hawthorne and Arthropod ABCB FT/P-gps have also been reported to be involved in Dively, 2011; Srinivas et al., 2005). colchicine transport, a secondary metabolite from plants of the genus Colchicum (autumn crocus) and a well-known human P-gp 4.2.5. Neonicotinoids, diflubenzuron and fipronil inhibitor (Seelig and Landwojtowicz, 2000). D. melanogaster mdr49 Only three studies have associated ABC transporters with deletion mutants showed increased sensitivity to colchicine (Wu neonicotinoid transport and/or resistance. Microarray gene et al., 1991) while Tapadia and Lakhotia (2005) showed that expression studies revealed that an ABCG transporter gene was colchicine induced mdr49 expression in D. melanogaster larvae. 106 W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110

More recently, the group of Aurade et al. (2010a, 2011) found that result in any phenotype with cuticle defects (Broehan et al., 2013). several secondary plant compounds are potent inhibitors of P-gp Together, these studies would argue against SUR as the target site of activity. A curcumin mixture, extracted from Turmeric (Curcuma BPU. In contrast, the processive glycosyltransferase chitin synthase longa) powder, inhibited H. armigera P-gp ATPase activity by 90% 1 (CHS1) has been shown to be the target site of etoxazole in the and inhibited TMR transport in proteoliposomes containing mite T. urticae. Although etoxazole is a diphenyl oxazoline acaricide H. armigera P-gp. In addition, it was found that H. armigera P-gp also that belongs to a different chemical family, the symptoms of showed large tryptophan fluorescence quenching in the presence poisoning strongly resemble those of BPUs in insects. Van Leeuwen of 10 mM of this curcuminoid mixture, suggesting that curcumi- et al. (2012) developed a population-level bulk segregant mapping noids may bind with ATP-binding sites and steroid acting regions. method to map a monogenic, recessive etoxazole resistance with Similar effects were also reported for the flavonoids morin, quer- high resolution to a single locus in the T. urticae genome. Additional cetin, and phloroglucinol (Aurade et al., 2011). Interestingly, genetic evidence further supported a mutation in a conserved C- H. armigera resistant larvae fed with diet supplemented with eth- terminal domain of CHS1, which is highly conserved and might ylparaoxon or cypermethrin (100 mMg 1 diet), insecticides known serve a non-catalytic but essential function. The authors suggest to stimulate H. armigera P-gp ATPase activity (see Sections 4.2.1 and that CHS1 may be a target site for BPUs in insects as well, although 4.2.4), and 500 mM morin or quercetin, showed a significant similar mutations as documented in T. urticae have not been re- reduction in weight gain and survival rate compared to H. armigera ported yet for any insect species. larvae that were fed with diet supplemented with either ethylparaoxon, cypermethrin, morin or quercetin alone (Aurade et al., 4.4.2. ABCC2 and Bt mode of action and resistance 2011). Furthermore, Petschenka et al. (2013) showed by immuno- Very recently, an ABC transporter (ABCC2) has been shown to be staining that an ABCB FT/P-gp transporter is expressed in the involved in resistance to insecticidal protein toxins from the bac- perineurium of the oleander hawkmoth, Daphnis nerii, and that terium B. thuringiensis (Bt toxins) (Heckel, 2012). The mode of ac- verapamil suppressed the efflux of the cardenolide digoxin from tion of Bt toxins (and the resistance mechanisms to Bt in insects) the perineurium in both D. nerii and M. sexta, suggesting that a are of huge commercial value, given that millions of hectares have ABCB FT/P-gp transporter mediates the efflux of digoxin. Finally, it been planted with transgenic Bt crops such as maize and cotton was found for the mountain pine beetle Dendroctonus ponderosae (Tabashnik et al., 2013). Independent studies in several insect that an ABCG transporter was upregulated in adults fed on lodge- species including H. virescens, T. ni, P. xylostella, have associated pole pine host tree tissue compared to adults that were starved mutations in ABCC2 with Bt resistance (Atsumi et al., 2012; Baxter (Robert et al., 2013). et al., 2011; Gahan et al., 2010), recently reviewed by Heckel (2012). The most convincing study by Atsumi et al. (2012) used germ line 4.4. ABC transporters as targets for insecticides transformation of the silkworm B. mori to provide functional evidence that a single tyrosine insertion in the extracellular loop of a 4.4.1. SUR and benzoylphenylureas TMD was sufficient to confer Bt resistance. Recently it was also Next to the transport of insecticides and allelochemicals, ABC shown, through ectopical expression of B. mori ABCC2 in Sf9 cells, transporters have also been studied as target sites for insecticides. that B. mori ABCC2 functions as a receptor for Cry1A toxins (Tanaka Probably the most well-known ABC transporter in arthropod toxi- et al., 2013). The mode of action of Bt toxins is a complex multi-step cology is the sulfonylurea receptor (SUR, ABCC family, see Section process, which involves activation of the pro-toxin by proteases 2.3 for detailed description), a conserved protein with orthologues after interaction with 12-cadherin domain proteins, formation of an in many invertebrate and vertebrate species including humans. In oligomeric pre-pore structure and several binding steps that arthropods, SUR has been put forward as the target site of ben- involve several midgut membrane proteins (see Bravo et al., 2011; zoylphenylureas (BPUs), a class of selective insecticides that inhibit Gahan et al., 2010). It has been proposed that ABCC2 plays a role chitin synthesis in insects and mites. The interference of BPUs with in the insertion of the pre-pore structure into the midgut mem- chitin synthesis was uncovered soon after their development and brane. The transporter in the open state is exposed to the outside of commercialization, as the lead compound diflubenzuron was the cell (see Section 1) and could interact with extended helices of shown to inhibit in vivo the incorporation of UDP-N-acetylglucos- the pre-pore Bt structure, followed by irreversible insertion into the amine (the building blocks of the chitin polymer) into chitin (Post membrane upon closure (Heckel, 2012). Given the interest in Bt and Vincent, 1973). However, all further attempts to document a resistance mechanisms, it can be expected that the precise mech- direct biochemical effect on any of the enzymes in the chitin syn- anism of this interaction will be the subject of extended future thesis pathway failed in the mid 1980s (Matsumura, 2010). The research. group of Matsumura has pioneered BPU mode of action studies and put forward the hypothesis of an interaction between BPUs and Acknowledgments SUR (reviewed in Matsumura, 2010). However, biochemical evidence of such interaction is still missing and counter arguments for T.V.L. and W.D. are postdoctoral fellows of the Fund for Scientific SUR as the target site of BPUs have recently been accumulating. Research Flanders (FWO). We thank Gunnar Broehan and Sheng Li fl First, although Drosophila is very sensitive to di ubenzuron, the for providing T. castaneum and B. mori ABC sequences, respectively. SUR mRNA is not detected in the epidermis where chitin synthesis takes place (Gangishetti et al., 2009). More convincingly, by taking Appendix A. Supplementary data advantage of genetic resources in Drosophila, Meyer et al. (2012) show that SUR is dispensable for chitin synthesis in Drosophila,as Supplementary data related to this article can be found at http:// larvae with completely eliminated SUR function display a normal dx.doi.org/10.1016/j.ibmb.2013.11.001. cuticular architecture. In addition, although the beetle T. castaneum is a model insect for studying the effects of chitin synthesis inhibitors such as diflubenzuron (Merzendorfer et al., 2012), a SUR References homolog has not been detected in this species or any other cole- Akiyama, M., 2013. The roles of ABCA12 in epidermal lipid barrier formation and opteran ((Broehan et al., 2013), see also Section 2.3). Furthermore, keratinocyte differentiation. Biochim. Biophys. Acta. http://dx.doi.org/10.1016/ RNAi knockdown of ABC transporters in T. castaneum did also not j.bbalip.2013.08.009. W. Dermauw, T. Van Leeuwen / Insect Biochemistry and Molecular Biology 45 (2014) 89e110 107

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