Open Access

Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.)

  • Weiping Qi1, 2, 3,
  • Xiaoli Ma1, 2, 3,
  • Weiyi He1, 2, 3,
  • Wei Chen1, 2, 3,
  • Mingmin Zou1, 2, 3,
  • Geoff M. Gurr1, 2, 4,
  • Liette Vasseur1, 5 and
  • Minsheng You1, 2, 3Email author
Contributed equally
BMC Genomics201617:760

https://doi.org/10.1186/s12864-016-3096-1

Received: 7 April 2016

Accepted: 16 September 2016

Published: 27 September 2016

Abstract

Background

ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins.

Results

A total of 82 ABC genes were identified from our published P. xylostella genome, and grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. Genes of subfamilies ABCA, ABCC and ABCH were found to be expanded in P. xylostella compared with those in Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens. Phylogenetic analysis indicated that many of the ABC transporters in P. xylostella are orthologous to the well-studied ABC transporter genes in the seven other species. Transcriptome- and qRT-PCR-based analysis elucidated physiological effects of ABC gene expressions of P. xylostella which were developmental stage- and tissue-specific as well as being affected by whether or not the insects were from an insecticide-resistant strain. Two ABCC and one ABCA genes were preferentially expressed in midgut of the 4th-instar larvae of a susceptible strain (Fuzhou-S) suggesting their potential roles in metabolizing plant defensive chemicals. Most of the highly expressed genes in insecticide-resistant strains were also predominantly expressed in the tissues of Malpighian tubules and midgut.

Conclusions

This is the most comprehensive study on identification, characterization and expression profiling of ABC transporter genes in P. xylostella to date. The diversified features and expression patterns of this gene family may be associated with the evolutionary capacity of this species to develop resistance to a wide range of insecticides and biological toxins. Our findings provide a solid foundation for future functional studies on specific ABC transporter genes in P. xylostella, and for further understanding of their physiological roles and regulatory pathways in insecticide resistance.

Keywords

ABC transporter Phylogenetic analysis Transcriptome analysis Insecticide resistance Detoxification

Background

ATP-binding cassette (ABC) transporters constitute one of the largest transmembrane protein families, which is widespread across all species. The first ABC transporter was found in prokaryotes, and the first cloned and characterized human ABC transporter member was ABCB1, which confers multidrug resistance (MDR) to cancer cells, preventing the accumulation of chemotherapeutic drugs [1]. According to their functions, the ABC proteins can be divided into three categories: importers, exporters and non-transport proteins [2]. Importers are only found in prokaryotes. In eukaryotes, ABC transporters are exporters and involved in the excretion of drugs, and endogenous and exogenous toxins. The third class of ABC proteins are apparently not related to molecule transport, but rather acting as ion channels, regulators of ion channels and receptors, and in some cases involved in DNA repair, ribosome assembly and translation [3].

ABC transporters are generally composed of four core domains with two being nucleotide-binding domains (NBDs) that bind and hydrolyze ATP and two transmembrane domains (TMDs) mediating translocation of the respective substrate [4]. Reflecting the diversity of substrates handled by ABC transporters, TMDs are much more diverse than NBDs, whose sequences are highly conserved in order to perform their roles as ATP hydrolyzing enzymes [5]. Half-transporters consist of one NBD and one TMD and need to form homo- or heterodimers to be functional [6]. The mode of action of ABC transporters is called “ATP-switch” transport cycle [7, 8]. The ABC family is classified into eight subfamilies, annotated A to H according to their sequence similarity and domain conservation [3]. The H subfamily appears to be present in all insects, mites, the slime mould Dictyostelium, and zebrafish, but is absent in genomes of plants, worms, yeasts, and mammalian species [9, 10]. In humans, many ABC proteins have been characterized with special functions and mutations of ABC genes can cause or contribute to a series of genetic disorders [11].

ABC transporters have recently been documented in insects as a family of detoxification-involved proteins [12], complementing the activity of another three classes of major metabolic enzymes: cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs) and carboxylesterases (COEs). Detoxification of toxins occurs in three phases with the first phase being associated predominantly with P450s [13]. In phase II, GSTs are dominant and known to be linked to resistance development to most classes of insecticides [14]. A series of transporters, including members of ABC transporters, are involved in phase III, which aims at the elimination of products generated during phases I and II [15]. Some ABC members of subfamilies B, C and G are involved in resistance to xenobiotics including insecticides [12]. Epis et al. [16] report that combining the insecticide permethrin with the ABC transporter inhibitor leads to greater Anopheles stephensi mortality than when using permethrin alone, demonstrating the importance of ABC transporters in insecticide resistance. Besides their detoxification roles, RNAi-mediated knockdown of some ABC genes in Tribolium castaneum results in a series of abnormal developmental phenotypes, such as growth arrest, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality [17].

The insect pest Plutella xylostella is a cosmopolitan Lepidoptera that almost exclusively feeds on cruciferous plants [18]. Due to its short life cycle and capacity to rapidly develop insecticide resistance, P. xylostella is difficult to control [19, 20]. The species is the first to be reported resistant to dichlorodiphenyltrichloroethane (DDT) in the 1950s [21] and Bacillus thuringiensis (Bt) toxins in the 1990s [22]. Bt resistance of P. xylostella is associated with ABCC2 alone [23] or in combination with ABCC3 [24] or ABCG1 [25]. In addition, the silencing of an ABCH1 gene results in the death of larvae and pupae [26]. Expression of ABC genes is found to be more frequently up-regulated than that of GSTs, COEs or P450s in insecticide-resistant larvae of P. xylostella, suggesting a potential detoxification role of ABC transporters [27].

In this study, based on the ABC transporter genes (PxABCs) previously identified from the P. xylostella genome [27], we further characterized the gene structure and motifs, and performed phylogenetic analysis using P. xylostella, Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae, and Homo sapiens to further understand the evolutionary relationships among the eight subfamilies identified in this study. In addition, we carried out transcriptome- and qRT-PCR-based expression profiling of the ABC transporter genes in different developmental stages, tissues, and insecticide-susceptible and resistant strains of P. xylostella.

Results and discussion

Identification and grouping of the PxABCs

Based on our previous work on annotation of PxABCs in the P. xylostella genome [27], we identified 82 ABC transporter genes (Table 1 and Additional file 1) and 19 ABC fragments (Additional file 2). The 19 ABC fragments had homology to ABC transporters of other insects, but lacked the highly conserved NBDs of canonical ABC proteins [4]. P. xylostella ABC transporter genes were grouped into the eight subfamilies (A-H) (Additional file 3). The number of genes in each subfamily greatly varied, ranging from one gene in ABCE to 21 in ABCC (Table 2). The ABCC subfamily was further divided into two groups with one group highly similar to the ABCB subfamily, which was also found in the other Lepidoptera, B. mori [28].
Table 1

Description of subfamily-based ABC transporter genes identified in the P. xylostella genome

Subfamily

Gene ID

Protein (aa)

Scaffold

Position

No. exons

RNA-seqa

qRT-PCRb

ABCA1

Px001174

969

scaffold_1135

304…19217

19

+

ABCA2

Px004981

1266

scaffold_188

295012…323041

28

+

ABCA3

Px004982

1186

scaffold_188

301796…321736

26

+

ABCA4

Px008069

1933

scaffold_288

24893…72425

42

+

ABCA5

Px008254

1086

scaffold_295

204319…225237

19

+

ABCA6

Px008255

739

scaffold_295

226197…247340

17

+

ABCA7

Px008256

1569

scaffold_295

250906…269981

29

+

ABCA8

Px009697a

233

scaffold_346

259984…265745

6

+

ABCA9

Px009697b

1296

scaffold_346

286234…303713

24

+

ABCA10

Px009783

1852

scaffold_35

644704…664586

37

+

ABCA11

Px013614

3796

scaffold_568

132102…192807

72

+

+

ABCA12

Px013659

786

scaffold_570

19302…30379

18

+

+

ABCA13

Px016911

1195

scaffold_856

62…22277

35

+

+

ABCA14

Px016912

2714

scaffold_856

24266…52676

49

+

ABCA15

Px017838

4008

scaffold_97

202450…263565

73

+

ABCB1

Px000163

1219

scaffold_10

1427959…1440157

22

+

ABCB2

Px002636

658

scaffold_14

2273580…2292376

18

+

ABCB3

Px002801

959

scaffold_142

423653…432357

17

+

ABCB4

Px004522

124

scaffold_175

1104754…1106729

3

+

ABCB5

Px005591

1257

scaffold_200

68221…86053

24

+

ABCB6

Px006391

862

scaffold_225

442449…454413

17

+

ABCB7

Px007221

1568

scaffold_254

255160…283670

30

+

ABCB8

Px007992

823

scaffold_284

306641…321481

18

+

ABCB9

Px008679

1218

scaffold_306

348764…361734

23

+

ABCB10

Px009649

912

scaffold_344

75019…96464

18

+

ABCB11

Px012211

1279

scaffold_479

179910…193122

23

+

ABCB12

Px013177

329

scaffold_53

1202314…1212357

7

+

ABCB13

Px013728

1308

scaffold_58

462696…478688

26

+

ABCB14

Px013729

1699

scaffold_58

487550…504266

31

+

ABCC1

Px001274

548

scaffold_1153

4862…13438

12

+

ABCC2

Px002416

1327

scaffold_49

2357…26234

2

+

+

ABCC3

Px002415

1406

scaffold_137

488447..513162

26

+

ABCC4

Px002418

912

scaffold_137

552867…576380

16

+

+

ABCC5

Px002419

471

scaffold_137

576715…581467

9

+

+

ABCC6

Px002696

1881

scaffold_140

577784…598125

27

+

ABCC7

Px002784

1069

scaffold_142

99127…105692

15

+

ABCC8

Px004042

1098

scaffold_164

402013…423822

22

+

ABCC9

Px005403

1274

scaffold_199

854564…879035

24

+

ABCC10

Px005931

143

scaffold_21

2289257…2290685

3

+

ABCC11

Px005932

451

scaffold_21

2289194…2309332

14

+

ABCC12

Px006710

1397

scaffold_236

4405…26723

24

+

ABCC13

Px008999

1818

scaffold_316

108977…138269

29

+

+

ABCC14

Px009134a

662

scaffold_321

178075…188823

14

+

ABCC15

Px009134b

861

scaffold_321

194897…231476

27

+

ABCC16

Px009834

1333

scaffold_352

169022…191602

24

+

+

ABCC17

Px009835

1262

scaffold_352

192939…220411

24

+

+

ABCC18

Px012780

891

scaffold_51

11388…27660

15

+

ABCC19

Px014427

806

scaffold_627

73391…97213

19

+

ABCC20

Px015447

710

scaffold_712

47919…65879

12

+

ABCC21

Px015888

211

scaffold_749

69505…71718

3

+

ABCD1

Px007715

700

scaffold_274

451324…468854

18

+

+

ABCD2

Px010231

765

scaffold_372

135778…153573

16

+

+

ABCD3

Px010704

619

scaffold_395

100478…120592

9

+

ABCE1

Px007660

267

scaffold_271

5031…10235

11

+

ABCF1

Px001241

901

scaffold_115

254134…267193

18

+

ABCF2

Px002158

621

scaffold_132

19709…32348

11

+

+

ABCF3

Px006766

657

scaffold_239

104170…121670

14

+

+

ABCG1

Px000087

575

scaffold_1

1687310…1700888

8

+

ABCG2

Px001524

646

scaffold_120

391766…407227

11

+

ABCG3

Px002116

141

scaffold_131

33576…47509

2

+

ABCG4

Px002117

490

scaffold_131

50160…54319

9

+

ABCG5

Px004725

583

scaffold_180

233059…240664

11

+

+

ABCG6

Px005467

518

scaffold_2

1210934…1215177

9

+

ABCG7

Px007185

587

scaffold_252

315812…338592

12

+

ABCG8

Px007949

1239

scaffold_282

234990…275031

22

+

ABCG9

Px007950

614

scaffold_282

282382…298666

12

+

ABCG10

Px008370

787

scaffold_3

1084031…1102351

15

+

ABCG11

Px008371

689

scaffold_3

1107251…1133659

14

+

+

ABCG12

Px012058

763

scaffold_47

714983…763358

12

+

ABCG13

Px016406

691

scaffold_8

151167…158192

12

+

ABCG14

Px016407

653

scaffold_8

190187…201753

13

+

ABCG15

Px016675

639

scaffold_82

554151…570599

10

+

ABCG16

Px016677

641

scaffold_82

593974…605475

10

+

+

ABCG17

Px016679

603

scaffold_82

593448…622103

14

+

ABCG18

Px017344

499

scaffold_909

2090…8847

10

+

ABCG19

Px017858

736

scaffold_97

716767…726144

7

+

ABCH1

Px003594

778

scaffold_158

256543…275266

16

+

ABCH2

Px004510

693

scaffold_175

841069…867340

12

+

ABCH3

Px005110

821

scaffold_19

1346939…1358906

13

+

ABCH4

Px005111

813

scaffold_19

1363123…1425269

15

+

ABCH5

Px014955

781

scaffold_677

19741…43381

14

+

+

ABCH6

Px014956

899

scaffold_677

45879…80000

17

+

a “+” represents the gene could be found in the P. xylostella transcriptome

b “+” represents the gene was validated by qRT-PCR, and “-” represents the gene was not validated

Table 2

Numerical distribution of subfamilies (A - H) based on ABC transporter genes of different species

Species

A

B

C

D

E

F

G

H

Total

Reference

Homo sapiens

13

11

12

4

1

3

5

0

48

[3]

Saccharomyces cerevisiae

0

4

6

2

2

6

10

0

30

[31]

Drosophila melanogaster

11

8

14

2

1

3

15

3

57

[3]

Tribolium castaneum

10

6

35

2

1

3

13

3

73

[17]

Apis mellifera

3

5

9

2

1

3

15

3

41

[28]

Daphnia pulex

4

7

7

3

1

4

23

15

64

[33]

Bombyx mori

6

8

15

2

1

3

13

3

51

[28]

Tetranychus urticae

9

4

39

2

1

3

23

22

103

[10]

Plutella xylostella

15

14

21

3

1

3

19

6

82

[26] and the present study

Manduca sexta

7

10

9

2

1

3

16

3

51

The present study

Danaus plexippus

8

16

12

3

1

3

16

3

62

The present study

Heliconius melpomene

10

11

15

2

1

3

17

3

62

The present study

Characterization of the PxABCs and their motifs

The 82 PxABCs were dispersed on 59 scaffolds, 40 of which were found being individually located on different scaffolds. The remaining PxABCs were clustered on 19 scaffolds with each containing two or three genes, suggesting tandem duplication of these genes. The length of most predicted ABC transporters ranged from 124 to 2,714 amino acids (aa), with two exceptionally long genes containing 3,796 and 4,008 aa. The corresponding exon numbers ranged from 2 to 73 (Table 1), showing high structural complexity (Additional file 3).

The NBDs of ABC transporters generally contain seven highly conserved, but not invariant, motifs including Walker A, Walker B, ABC signature, A-loop, Q-loop, D-loop and H-loop [8]. The Walker motifs A and B show that the ATP binding sites [29] and the “signature sequence” (also called C-loop or LSGGQ motif) are exclusive to ABC transporters, contributing to the formation of a composite catalytic site [5]. Additional motifs such as the A-loop, Q-loop, D-loop and H-loop (also called the switch loop) only possess a single conserved aa residue [5], which are key amino acids in the catalytic cycle. We identified in P. xylostella six of these seven conserved motifs, with A-loop being absent (Fig. 1). However, the nature of such structural invariance in NBDs among arthropods remains poorly understood [30].
Fig. 1

Illustration of six conserved motifs identified from the P. xylostella ABC transporters. The overall height of each column is proportional to the information content of all of the amino acids at that position, and within each of the columns the conservation of each residue is visualized as the relative height of symbols representing amino acids. Walker A, GxxGxGKST; Walker B, LLDEPT; D-loop, LD; ABC signature, LSGGQ; H-loop, xHx; Q-loop: xQx

Subfamily-based comparison of ABC transporters

ABCA

In P. xylostella, 15 ABCA genes were identified, including one 3-gene cluster on scaffold 295, two 2-gene clusters on scaffolds 188 and 856, and eight genes that were individually located on different scaffolds. Apparently, gene tandem duplication resulted in high protein diversity of ABCA. ABCA subfamily harbored the two longest ABC transporter genes, Px013614 (3,796 aa) and Px017838 (4,008 aa), which were similar in length to B. mori [28] and T. urticae [10]. In Saccharomyces cerevisiae (yeast), no ABCA member has been identified [31] (Table 2). ABCA transporters in human are characterized as full-transporters [3], however, we found their structures are variable in arthropods. The ABCA transporters of T. urticae and D. melanogaster were all full-transporters, while the moths and butterflies contained both full- and half-transporters (Additional file 4). The P. xylostella ABCA subfamily comprised of nine full- and six half-transporters. Phylogenetic analysis of the ABCA subfamily revealed a Lepidoptera-specific clade with a distinct expansion in P. xylostella, which was orthologous to a human-specific clade (Fig. 2). A same orthologous relationship was also observed between another Lepidoptera-specific clade and a mite-specific clade (Fig. 2).
Fig. 2

Phylogenetic analysis of ABCA transporters of eight species. Full-length ABCA proteins among eight species were aligned using MUSCLE, and subsequently to generate a phylogenetic tree using a maximum likelihood analysis with 1000 replications. Species are differentially coded with Hs for H. sapiens, Tu for T. urticae, Dm for D. melanogaster, Dp for D. plexippus, Hm for H. melpomene, Bm for B. mori, Ms for M. sexta and Px for P. xylostella. The branches with bootstrap support > 80 % were dotted in orange, and the ones harboring PxABCs were in red. Protein sequences used for phylogenetic analysis are provided in the Additional file 4

ABCA transporters in human are involved in lipid transport and metabolism [3]. For example, the ABCA1 protein is mutated in a recessive disorder (Tangier disease) characterized by a defect in biogenesis of high density lipoprotein (HDL) [32]. It is reported that HsABCA4 is linked to the Stargardt disease because of its role in retinal integrity, HsABCA3 is involved in lung surfactant production, and HsABCA12 participates in keratinization processes in the skin [11]. However, the roles of ABCA transporter genes in arthropods are currently unclear. They may share a function related to lipid trafficking processes based on the high conservation of their structure. RNAi-mediated knockdown of TcABCA-9A or TcABCA-9B results in ~30 % mortality of T. castaneum [17], implying their significant roles in insects.

ABCB

ABCB subfamily contains both full- and half-transporters [33]. In the P. xylostella genome, we identified seven full- and seven half-transporters. The phylogenetic analysis of full-transporters showed that H. sapiens and T. urticae were located in a separate clade from insects (Fig. 3a). The phylogenetic tree of most ABCB half-transporters showed obvious orthologs and high bootstrap values among the eight species (Fig. 3b), indicating that they were orthologous and evolutionary divergent. Our results suggest that full-transporters may have evolved through lineage-specific duplication, while half-transporters may be evolutionarily conserved in metazoan species [10, 28, 33].
Fig. 3

Phylogenetic analysis of full- (a) and half- (b) transporters in the ABCB subfamily of eight species. See the legend of Fig. 2 for performance and presentation details. Protein sequences used for phylogenetic analysis are provided in the Additional files 5 (full) and 6 (half)

In human, the full-transporter ABCB1 plays important roles in cancer development by contributing to MDR [1]. Expression of the ABCB1 gene of P. xylostella, PxPgp1, was found being up-regulated in the strain subjected to insecticide abamectin [34]. In Heliothis virescens, increased expression of the human Pgp orthologs is associated with resistance to thiodicarb [35]. Mutations in bile salt export pump gene (BSEP, HsABCB11) and multidrug-resistant gene 3 (MDR3, HsABCB4) in human can result in progressive familial intrahepatic cholestasis [36]. Based on the phylogenetic tree (Fig. 3a), HsABCB4 and HsABCB11, both being full-transporters, seemed to be functioning in a human-specific manner. Human ABCB6, 7, 8 and 10 are mitochondrial half-transporters involved in iron metabolism and transport of Fe/S protein precursors [37]. Their orthologs in arthropods are clear (Fig. 3b), implying the similar roles as in humans [30]. However, the human-specific ABCB half-transporters (Fig. 3b), ABCB2, 3 and 9, are involved in antigen processing [37].

ABCC

In P. xylostella, ABCC transporters formed the largest subfamily with 21 members (Table 1). There were one 3-gene cluster on scaffold 137, two 2-gene clusters on scaffolds 21 and 321, and 14 genes that were individually located on different scaffolds, suggesting tandem duplication of those clustered genes. Human ABCC transporters are all full-transporters, but in insects both full- and half-transporters can be found [3, 15, 28]. It showed similar structural characteristics to what we have found in ABCA subfamily of the eight species. The P. xylostella ABCC subfamily consisted of eight full- and 13 half-transporters. The phylogenetic tree of ABCC transporters showed that lineage-specific members were significantly duplicated in T. urticae, while this kind of duplication was inconspicuous in other species, suggesting varying evolutionary divergence of ABCC subfamily among the studied species (Fig. 4).
Fig. 4

Phylogenetic analysis of ABCC transporters of eight species. See the legend of Fig. 2 for performance and presentation details. Protein sequences used for phylogenetic analysis are provided in the Additional file 7

ABCC transporters are regarded as multidrug-resistance associated proteins (MRPs) due to their ability to extrude drugs with broad specificity. In D. melanogaster adults, MRPs may play a role in secretion of methotrexate by the Malpighian tubules [38], which is a drug used for treating cancers and auto-immune disorders [39]. Labbe et al. propose that ABCC transporters can be involved in xenobiotic efflux of moths [12]. Similar to ABCB4, ABCC2 is also involved in bile acids, phospholipids, and bilirubin export and related to progressive familial intrahepatic cholestasis [36]. ABCC7 (cystic fibrosis transmembrane conductance regulator, CFTR) is an important ABC transporter involved in ion transport in human, and those who have mutations on functional CFTR may suffer diseases [40, 41]. However, no orthologs of human ABCC7 were found in P. xylostella and the other arthropod species.

Although mutations in ABCC2 have been implicated in Bt resistance in H. virescens, P. xylostella, Trichoplusia ni, and B. mori [23, 4244], species-specific patterns of cross-resistance to Bt toxins are varied. This suggests that different mutations in ABCC2 may have occurred influencing the types of Bt resistance in a toxin binding site-dependent manner [45]. Recently, ABCC3 ortholog in P. xylostella (Px008999) has been reported to also be associated with Bt resistance in insects [24], while its ortholog in human (ABCC3) functions as a marker for MDR in non-small cell lung cancer [46].

ABCC transporters also have cell-surface receptor activity, such as sulfonylurea receptor (SUR) [47]. Among the eight species studied, P. xylostella and M. sexta had no ortholog of SUR (Fig. 4). In arthropods, SUR has been proposed as the direct target site for benzoylphenylureas (BPUs), a group of chitin synthesis inhibitors [48]. The insecticidal activity of BPUs in P. xylostella [49] provides a clue for further exploring its SUR gene. Recent research however suggests that SUR may not be the only receptor for chitin synthesis as shown in D. melanogaster embryos [50]. Knocking down SUR ortholog in T. castaneum has no effect on chitin synthesis [17].

ABCD

The ABCD subfamily solely consists of half-transporters with the topology TMD–NBD, except for some plant representatives [47]. All three ABCD members in P. xylostella were half-transporters. ABCD subfamily was present in all studied taxa and comprised of two to five members (Table 2). The ABCDs showed four distinct groups with HsABCD4 standing in its own branch and the three PxABCDs being clustered into two Lepidoptera-specific clades (Fig. 5). Their high orthologous relationships indicate that they are evolutionarily conserved in metazoan species.
Fig. 5

Phylogenetic analysis of ABCD transporters of eight species. See the legend of Fig. 2 for performance and presentation details. Protein sequences used for phylogenetic analysis are provided in the Additional file 8

ABCD transporters are peroxisomal membrane-located and are involved in importing fatty acids and/or fatty acyl-CoAs into peroxisome with specific metabolic and developmental functions in various organisms [51]. However, the role of ABCD transporters in arthropods is still unclear [30].

ABCE and ABCF

ABCE proteins showed a high similarity (>50 % of aa identity) among the studied species (Fig. 6). The numbers of ABCE and ABCF were highly conserved, with most eukaryotes (including P. xylostella) having one ABCE and three ABCF genes (Table 2). Phylogenetic analysis showed that they had clear orthologous relationships (Fig. 6).
Fig. 6

Phylogenetic analysis of ABCE and ABCF transporters of eight species. See the legend of Fig. 2 for performance and presentation details. The branches that harbor ABCE genes were presented in dotted line. Protein sequences used for phylogenetic analysis are provided in the Additional files 9 (ABCE) and 10 (ABCF)

The structure of ABCE and ABCF are quite distinct from other ABC transporters due to their lack of TMDs and only containing a pair of linked NBDs [52]. Therefore, they are not related to transport. Initially, ABCE was identified as an RNase L inhibitor in human [53]. In human and yeast, ABCE proteins contribute in translation initiation [54]. ABCF is similar to ABCE and relates to ribosome biogenesis and translational control. RNAi-mediated knockdown of members in the ABCE and ABCF subfamilies in the flour beetle T. castaneum results in 100 % mortality in penultimate larvae, showing fundamental roles of ABCE and ABCF in biological processes of the insects [15]. In P. xylostella, the ABCE gene (Px007660) exhibited the highest expression level compared to other PxABCs based on the P. xylostella genome [27] and transcriptome [55]. The same result is also reported in B. mori [28].

ABCG

In P. xylostella, we found a total of 19 ABCG transporters, with 18 of them being half-transporters and only one full-transporter (Px007949). Gene duplication appeared to have occurred several times among ABCG genes of P. xylostella, which was evidenced by the location of gene paralogs on scaffolds 3, 8, 82, 131 and 282 (Table 1). Most ABCG transporters are half-transporters and they need to form homo- or hetero-dimers to perform the transport function [30].

In the phylogenetic tree of ABCG, all the orthologs of Dmwhite, Dmbrown and Dmscarlet were identified in Lepidoptera (Fig. 7). They are the most well-known ABCG genes in D. melanogaster, and encode for proteins to transport guanine or tryptophan precursors of the red and brown eye color pigments influencing the development of compound and simple eyes [56, 57]. These orthologs are also present in other insects, such as Anopheles gambiae [58], Bactrocera dorsalis [59] and Ceratitis capitata [60], suggesting that white, brown and scarlet are highly conserved protein transporters for eye color pigments in insects. Although several P. xylostella ABCG proteins have similar sequences with the white, brown and scarlet proteins in D. melanogaster, it is unclear whether they are involved in eye color pigments. It has been reported that Pxwhite is associated with Bt resistance in P. xylostella [25]. In A. stephensi, analysis shows that the ortholog of Dmscarlet, AnstABCG4, exhibits a ten-fold increased expression when treated with permethrin after 48 h compared to untreated control [16].
Fig. 7

Phylogenetic analysis of ABCG transporters of eight species. See the legend of Fig. 2 for performance and presentation details. Protein sequences used for phylogenetic analysis are provided in the Additional file 11

In human, ABCG proteins are involved in lipid transport across membranes. The most intensively studied ABCG in human is ABCG2 (breast cancer resistance protein, BCRP), which acts as a MRP transporting anticancer drugs and a series of substrates [6163]. In our study, no human ABCG2 orthologs were identified in Lepidoptera (Fig. 7), which were identified in D. melanogaster (E23) and T. urticae. D. melanogaster E23 (DmCG3327) is a 20-hydroxyecdysone (20E) primary response ABC transporter, which can suppress 20E-mediated gene activation [64]. In B. mori, five ABCG genes including BmABC005226, BmABC005203, BmABC005202, BmABC010555 and BmABC010557, are 20E responsive genes although they are not orthologous to E23 [28]. The orthologous relationships of HsABCG5 and HsABCG8 with the other species showed their evolutionarily conserved function (Fig. 7), the translated proteins of which form a functional heterodimer that is coordinately regulated by cholesterol [3].

ABCH

We identified 6 ABCH genes in P. xylostella and they were all half-transporters. One of them (Px004510) tended to share high sequence similarity with ABCF subfamily (Additional file 3). ABCH subfamily was first identified with the sequencing of D. melanogaster genome [3]. The structure of ABCH proteins is most closely related to subfamily ABCG. A previous study using five insect species with available genomes suggests that insect ABCHs may have originated from a common ancestral copy [28]. Our phylogenetic analysis further supports this hypothesis (Fig. 8). Outside the Insecta, however, the T. urticae ABCHs seem not to conform to this rule (Fig. 8).
Fig. 8

Phylogenetic analysis of ABCH transporters of seven arthropod species. See the legend of Fig. 2 for performance and presentation details. Protein sequences used for phylogenetic analysis are provided in the Additional file 12

Down-regulation of an ABCH1 gene can cause high mortality of larvae and pupae of P. xylostella [26]. Similarly, RNAi-mediated knockdown of TcABCH-9C results in desiccation and 100 % mortality in T. castaneum [17], and knockdown of ABCH gene CG9990 in D. melanogaster is lethal [65, 66]. It may be inferred that ABCH is a newly evolved subfamily with specific functions in the development of certain species, but this needs to be investigated.

Stage- and tissue-specific expression of the PxABCs

In order to develop an understanding of the physiological functions of ABC transporters in P. xylostella, the expression of 82 PxABCs was profiled at different developmental stages and with different tissues of the susceptible strain (Fuzhou-S, SS) (Fig. 9 and Additional file 13), based on the P. xylostella genome and transcriptome datasets [27, 55]. The RPKM values of the 82 ABC genes were clustered into three separate clades. The first clade contained 22 ABC genes with higher expression levels in various tissues and developmental stages, including two ABCDs, one ABCE and two ABCFs, which showed particularly high expression levels. This indicated that while ABCD, ABCE and ABCF were numerically small subfamilies (Table 2), they might be of some biological importance, though currently unclear. Within the second clade, there were 25 ABC genes exhibiting low expressions with most RPKM values being > 1.
Fig. 9

Expression patterns of the ABC genes in multiple developmental stages and tissues of P. xylostella based on RPKM values. The relative expression levels are illustrated by seven scaled colors and corresponding log2 RPKM values (Additional file 13) ranging from −3.00 to +3.00. Tissue-specific expression profiling was performed using multiple tissues (if not specified) of the 4th larvae as shown on the top of the figure. The colors vary from bright red showing up-regulated expression to bright purple for down-regulated genes

The third clade contained 35 ABC genes with variable expression patterns. We found that Px002415 (ABCC3), Px002416 (ABCC2) and Px008256 (ABCA7) were relatively highly expressed in midgut of 4th instar and all larval stages compared with the other tissues and stages. Previous studies have shown that ABCC2 proteins play important physiological roles in resistance to Bt toxins [23, 42, 43]. Since midgut is an important organ involved with food digestion and detoxification, and larva is the main feeding stage of P. xylostella, we suspect that these genes may also play important roles in metabolizing plant secondary compounds. Many ABC genes were preferentially expressed in Malpighian tubule and most of them belonged to ABCB and ABCC subfamilies (Fig. 9). This suggests that ABC transporter genes of these subfamilies may also be involved in xenobiotic detoxification because Malpighian tubules make up an important excretory organ for transporting wastes [15].

Strain-specific expression profiles of the PxABCs

In order to understand the potential function of the P. xylostella ABC proteins in insecticide resistance, expressions of the P. xylostella ABC genes were profiled for larvae of two insecticide-resistant strains (fipronil (FRS) and chlorpyrifos (CRS)) (Fig. 10 and Additional file 14), based on the genome and transcriptome data and compared with the susceptible strain (SS) [27, 55]. The majority of the P. xylostella ABC genes were up-regulated in FRS and/or CRS, with a few genes being down-regulated. The ABCE gene (Px007660) showed the highest expression level in all three strains, suggesting its fundamental role in various physiological processes of the cell. At least, five ABCC genes including Px002418 (ABCC4), Px008999 (ABCC13), Px009835 (ABCC17), Px002416 (ABCC2), and Px002419 (ABCC5) were up-regulated in FRS and CRS. Among them, ABCC genes are proposed to be involved in insecticide resistance [12], and ABCC2 is linked to Bt resistance [23].
Fig. 10

Strain-specific expression patterns of the ABC genes in the 3rd instar larvae of P. xylostella based on RPKM values. The relative expression levels are illustrated by seven scaled colors and corresponding log2 RPKM values (Additional file 14) ranging from −2.00 to +2.00. SS: susceptible strain; CRS: chlorpyrifos-resistant strains; FRS: fipronil-resistant strain

In our case, one of the ABCH genes (Px014955) was mostly up-regulated in FRS and CRS, and weakly expressed in SS. Four genes including Px002784 (ABCC7), Px013614 (ABCA11), Px013659 (ABCA12) and Px015888 (ABCC21) showed up-regulated expression in CRS, but not in FRS, suggesting that the expression of these genes could be induced by specific insecticides. There were eight PxABCs exhibiting low expression (RPKM value < 1) in all three strains. Three of the PxABC genes (ABCA14, ABCB4 and ABCG4) had no differential expression in the three strains, suggesting that they might not be related to the resistance of chlorpyrifos and fipronil. Px006766 (ABCF3) and Px013659 (ABCA12) were down-regulated in FRS and CRS, while Px013614 (ABCA11), Px008371 (ABCG11) and Px004725 (ABCG5) exhibited up-regulated expression in both insecticide-resistant strains. This may indicate that not all ABC transporter genes are responsible for xenobiotics excretion, and a strictly transcriptional regulation of ABCs in response to external challenges is required for balancing transmembrane substrate transport.

qRT-PCR validation of expressions

Eighteen ABC transporter genes with up- or down-regulated expressions in the two insecticide-resistant strains (FRS and CRS) of P. xylostella were selected for experimental validation using qRT-PCR. Quantitative expression of the PxABCs at different developmental stages of the susceptible strain (Additional file 15) and two insecticide-resistant strains (Additional file 16) overall confirmed the stage- and strain-specific expression patterns as profiled using the P. xylostella genome and transcriptome datasets (Figs. 9 and 10). The results provide significant clues for further studies on functions of some specific PxABCs in host plant adaptation and insecticide resistance development.

Conclusions

Our work presents the most comprehensive study to date on identification, characterization and expression profiling of ABC transporters in the genome of P. xylostella, integrating evolutionary and physiological aspects. A comparison of ABC transporters from seven arthropod species and human provides an overview of this vital gene family. The variances in genomic features and expression patterns of the genes reflect evolutionary and functional diversification of the ABC transporters. Some genes are preferentially expressed in larvae, midgut, and Malpighian tubules, which may be involved in detoxification of plant defense chemicals. Moreover, some of the ABC transporter genes are up-regulated in two insecticide-resistant strains compared to the susceptible strain, suggesting their involvement in detoxification of the chemical insecticides through transportation. Our work offers a solid foundation for further research on gene-specific functions of the ABC transporters, which is essential to better understand the molecular and genetic mechanisms involved in detoxification in P. xylostella.

Methods

Experimental DBM strains

The experimental population of P. xylostella was derived from a susceptible strain (Fuzhou-S) that was collected from a vegetable field of Fuzhou (26.08°N, 119.28°E) in 2004 and used for genome sequencing [27]. Since then this initial population has been reared on potted radish seedlings (Raphanus sativus L.) at 25 ± 1 °C, 65 ± 5%RH and L:D = 16:8 h in a separate greenhouse without exposure to insecticides. The two insecticide-resistant strains (CRS and FRS) were selected from this susceptible strain, and detailed in the published P. xylostella transcriptome [55].

Identification of lepidopteran ABC transporters

To identify the ABC transporter genes in the P. xylostella genome (http://iae.fafu.edu.cn/DBM/), a systematic BLASTP search was performed using arthropod ABC transporter protein sequences available from NCBI as queries, with the cutoff set at e-value < e−20. Candidate ABC transporter sequences were submitted to the NCBI protein database to search for ABC transporter domains. We used the online FGENESH and FGENESH+ programs (http://linux1.softberry.com/berry.phtml) to predict the gene structures of ABC transporter genes on their genomic DNA sequences. Finally, we used program Pfam (http://pfam.xfam.org/) to identify the NBD and TMD structure. The same method was applied to identify ABC transporters from up to date genome versions of Manduca sexta (ftp://ftp.bioinformatics.ksu.edu/pub/Manduca/OGS2/), Danaus plexippus (http://monarchbase.umassmed.edu/resource.html), and Heliconius melpomene (http://www.butterflygenome.org/node/4).

Sequence alignment and phylogenetic tree construction

To assign the ABC transporter genes to specific subfamilies, the complete aa of NBDs of all P. xylostella ABC transporters were aligned by MUSCLE algorithm. Phylogenetic trees were constructed with the Maximum Likelihood method using MEGA-CC (7.0.18) for Linux users. Bootstrap analysis with 1,000 replicates was used to evaluate the significance of the nodes. Poisson correction aa model and all sites were used for the tree reconstruction. Comparison analyses were conducted among P. xylostella, B. mori, M. sexta, H. melpomene, D. plexippus, D. melanogaster, T. urticae and H. sapiens for each of the ABC transporter subfamilies separately using the full-length aa sequences and the same method as what phylogenetic analysis of P. xylostella ABC transporters used.

Examination of gene structure and motifs

The P. xylostella ABC genes were mapped onto corresponding scaffolds of the genome sequence assembly version 2 [27]. Gene structure of the P. xylostella ABC genes was visualized using the online tool Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). We used the MEME software (http://meme.sdsc.edu) to identify the conserved motifs in the 82 PxABC sequences using the following parameters: number of repetitions = any, maximum number of motifs = 10, and optimum motif width = 3 to 10 residues.

Gene expression profiling of the PxABCs

The RNA-seq data of the P. xylostella ABC genes were downloaded from the published database (http://iae.fafu.edu.cn/DBM/). Expressions of the PxABCs were profiled in different developmental stages and tissues of the susceptible strain, including eggs (within 24 h after oviposition), 1st ~ 4th-instar larvae, pupae, male and female adults, fat body, hemolymph, Malpighian tubules, silk glands, salivary glands and midgut of the 4th-instar larvae, head of the 4th-instar larvae and male/female adults, and individuals between 3rd-instar larvae of SS, and each of the insecticide-resistant strains (FRS and CRS). Each of the RPKM values was transformed into base-2 logarithm, and the expression profiling of ABC transporter genes was generated and visualized by pheatmap package of the R program (https://www.r-project.org/, version 3.2.5) using the similarity metric of Euclidean distance and clustering method of complete linkage.

Validation of the gene expression by qRT-PCR

To confirm expression patterns of the PxABCs based on P. xylostella transcriptome, qRT-PCR analysis was performed using SYBR-green fluorescence with gene-specific primers. The PCR products were examined by dissociation curve analysis after the PCR reaction to confirm the specific detection of target transcripts by the qRT-PCR analysis. Three independent biological replicates were included for qRT-PCR, each of which had three technical replicates. The first-strand cDNA was synthesized from total RNA using the reverse transcriptase kit from Promega (Madison, WI). The qRT-PCR reactions were prepared using the SYBR SELECT MASTER MIX FOR CFX from Invitrogen (Carlsbad, CA) following manufacturer’s instructions and run on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA), following the program: 95 °C for 3 min; 45 cycles of 95 °C for 15 s, and 57 °C for 35 s, and a final melt curve at 60 °C for 5 s to 95 °C with 0.5 °C increments. The P. xylostella ribosomal protein L32 (RPL32) gene (GenBank acc. no. AB180441) was used as an internal reference. Standard curves were generated by 5-fold dilutions of the cDNA templates. The 2−ΔCt method was used to analyze the relative values of mRNA expression.

Abbreviations

20E: 

20-hydroxyecdysone

aa: 

Amino acids

ABC: 

ATP-binding cassette

AnstABC: 

ABC transporter of A. stephensi

BCRP: 

Breast cancer resistance protein

BmABC: 

ABC transporter of B. mori

BPU: 

Benzoylphenylurea

Bt: 

Bacillus thuringiensis

CFTR: 

Cystic fibrosis transmembrane conductance regulator

CRS: 

Chlorpyrifos-resistant strains

DDT: 

Dichlorodiphenyltrichloroethane

FRS: 

Fipronil-resistant strain

HsABC: 

ABC transporter of H. sapiens

MDR: 

Multidrug resistance

MRP: 

Multidrug-resistance associated protein

NBD: 

Nucleotide-binding domain

PxABC: 

ABC transporter of P. xylostella

PxPgp1: 

P-glycoprotein 1 of P. xylostella

qRT-PCR: 

Quantitative real-time polymerase chain reaction

RPKM: 

Reads per kilobase per millionread

SS: 

Susceptible strain

SUR: 

Sulfonylurea receptor

TcABC: 

ABC transporter of T. castaneum

TMD: 

Transmembrane domain

Declarations

Funding

The work was supported by National Natural Science Foundation of China (Nos. 31320103922, 31230061 and 31301677), National Key Project of Fundamental Scientific Research (“973” Programs, No. 2011CB100404) in China, Natural Science Foundation of Fujian Proince, China (2014 J01086), and Outstanding Youth Fellowships for WH at FAFU (xjq201403). GMG is supported by the National Thousand Talents Program in China and the Advanced Talents of SAEFA, and LV by the Minjiang Scholar Program in Fujian Province (PRC) and the Advanced Talents of State Administration of Foreign Experts Affairs (PRC).

Availability of data and materials

All sequences used for this study are available in respective public databases and provided in additional supporting files. The phylogenetic data for Figs. 2, 3, 4, 5, 6, 7 and 8 were uploaded to TreeBase and the original NEXUS files can be downloaded from the DBM-DB (http://iae.fafu.edu.cn/DBM/family/PxABCs.php).

Authors’ contributions

WQ, WC and MZ designed and/or performed experiments. WQ drafted the manuscript. WQ, XM and WH carried out the bioinformatics analysis. MY supervised the project. MY, WQ, XM, WH, GMG and LV interpreted the data and critically revised the manuscript. All authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

P. xylostella is not protected under any legislation in China, as a protected or endangered species regulating or restricting its collection. Neither specific permits are required for collecting the specimens from the field nor is animal ethics approval required for work with this invertebrate.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Applied Ecology and Research Centre for Biodiversity and Eco-Safety, Fujian Agriculture and Forestry University
(2)
Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University
(3)
Key Laboratory of Integrated Pest Management of Fujian and Taiwan, China Ministry of Agriculture
(4)
Graham Centre, Charles Sturt University
(5)
Department of Biological Sciences, Brock University

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