Identification of candidate ATP-binding cassette transporter gene family members in Diaphorina citri (Hemiptera: Psyllidae) via adult tissues transcriptome analysis

The ATP-binding cassette (ABC) transporters exist in all living organisms and play major roles in various biological functions by transporting a wide variety of substrates across membranes. The functions of ABC transporters in drug resistance have been extensively studied in vertebrates; however, they are rarely characterized in agricultural pests. The Asian citrus psyllid, Diaphorina citri, is one of the most damaging pests of the Citrus genus because of its transmission of Huanglongbing, also known as Yellow Dragon disease. In this study, the next-generation sequencing technique was applied to research the ABC transporters of D. citri. Fifty-three ABC transporter genes were found in the RNA-Seq data, and among these ABC transporters, 4, 4, 5, 2, 1, 4, 18 and 15 ABC proteins belonged to the ABCA-ABCH subfamilies, respectively. Different expression profiles of 52 genes between imidacloprid-resistant and imidacloprid-susceptible strains were studied by qRT-PCR; 5 ABCGs and 4 ABCHs were significantly upregulated in the imidacloprid-resistant strain. In addition, five of the nine upregulated genes were widely expressed in adult tissues in spatial expression analysis. The results suggest that these genes may play key roles in this phenotype. In general, this study contributed to our current understanding of D. citri resistance to insecticides.


Results and Discussion
Identification of ABC transporters in D. citri. A total of 53 ABC transporter genes were found in the transcripts of D. citri, including 52 unigenes with full-length open reading frame (ORF) sequences and the lengths of these ABC transporters ranged from 594 to 2413 amino acids (Table 1). In addition, 91 ABC transporter genes or fragments were also found in the genome of D. citri (Accession, GCA_000475195.1), and all these genes or fragments can be matched with those identified from the transcriptome data (Table S2). Therefore, we speculate that the 53 ABC transporters we identified are very close to representing all of the ABC transporters. Then, we aligned the NBDs by the ClustalW program and constructed a neighbor-joining tree. According to the homology of the NBDs, these 53 ABC transporters were grouped into the 8 A-H families (Fig. 1). We identified 4, 4, 5, 2, 1, 4, 18 and 15 ABC proteins belonging to the ABCA-H subfamilies, respectively. All genes of subfamilies ABCA and ABCC were full-transporters; in subfamily ABCB, full-transporters were not identified. The ABCD, ABCG, and ABCH subfamilies comprised only half-transporters. However, subfamilies ABCE and ABCF contained only NBDs (Fig. 2). All of the ABC transporter genes of D. citri were submitted to GenBank (Table 1). ABCA subfamily. Four ABCA transporters were identified in D. citri. Three of them were full-transporters, and one was a single TMD-containing ABCA protein (DcABCA2) (Fig. 2). This subfamily includes the largest ABC transporter, which is encoded by DcABCA1 (2413 amino acids), and in fact, subfamily ABCA transporters are typically the largest among known ABCs 26 . A phylogenetic tree was constructed to support the member position for the DcABCAs (Supplementary Fig. S1). DcABCA1 clustered with tetur25g01640 and human HsABCA1, 2, 4 and 7. DcABCA2 reveals a high bootstrap support with a group that is formed by six ABCAs of B. tabaci, and then aligned with two ABCAs of T. castaneum, TcABCA-UA and TcABCA-7A, DmCG31731, BmABC004187 and a sister-group that is formed by seven T. urticae ABCAs; these ABCAs form a clade. DcABCA3 is placed in the human ABCA3 clade, which comprises a sister-group of TcABCAs. The clade also contains two BmABCAs (BmABC007217 and BmABC007221). DcABCA4 clustered with three insect ABCAs and then aligned with five human HsABCAs (HsABCA5, HsABCA6, and HsABCA8-10).
In humans, ABCA transporters play important functions in lipid transport and metabolism 2 . This includes ABCA1, which transports intracellular cholesterol and phosphatide to lipid-poor apolipoprotein A-I (ApoA-I) to form high-density lipoprotein (HDL) 27 . The expression of ABCA1 in the hippocampus is positively associated with the severity of Alzheimer's disease (AD) 28 . However, the role of the arthropod orthologues of these human ABCAs is currently unclear, but they might be related to lipid transport based on the high conservation of the structure. Injection of dsRNAs of TcABCA-9A and TcABCA-9B, results in approximately 30% mortality with severe defects in pupae and pharate adults of T. castaneum 29 . ABCB subfamily. The ABCB subfamily contains both full-transporters and half-transporters. In D. citri, four ABCB transporters were identified, and all of them are half-transporters that comprise one TMD and one NBD. In the phylogenetic tree ( Supplementary Fig. S2   three other clades similar to DcABCB2. The half transporters in phylogenetic analysis showed obvious orthologous relationships, suggesting that half-transporters have evolutionarily conserved roles in arthropods 30 . In humans, HsABCB6-8 and HsABCB10 are four mitochondrial ATP-binding cassette transporters. HsABCB6 is associated with multiple cellular functions, including iron homeostasis and porphyrin transport, and is resistant to several cytotoxic agents 31 . HsABCB7 is associated with Refractory anaemia with ring sideroblasts (RARS) 32 . HsABCB8 is involved in protecting the mitochondrial genome through doxorubicin resistance 33 . HsABCB10 is an important player in the protection from oxidative stress 34 . D. melanogaster DmCG4225, a homologous gene of Homo sapiens HsABCB6 was associated with tolerance to cadmium 35 . DmCG3879 (MDR49) is involved in directing germ cell migration through controlling export of a Drosophila germ cell attractant in a signal peptide-independent manner 36 . The homologous gene of HsABCB7 in Aedes aegypti is involved in insecticide resistance 37 .
ABCC subfamily. The C subfamily ABC transporters in humans consist of cystic fibrosis transmembrane conductance regulator (CFTR), membrane-bound sulfonylurea receptors (SURs) and multidrug resistance-associated proteins (MRPs). CFTR (HsABCC7) acts as a chloride channel that is involved in regulating exocrine secretions. SURs (HsABCC8, HsABCC9) binds sulfonylurea and functions as regulators of potassium channels that play a role in modulating insulin secretion. MRPs (HsABCC1-6 and HsABCC10-12) are considered to be important transporters of xenobiotics due to their ability to transport a wide range of substrates (such as drugs, ions, toxins, and endogenous compounds) 2,38,39 . Due to their functions, MRPs are the most well characterized in the ABC transporters subfamily C. All of the human ABCC transporter genes encode full ABC transporters; however, both full-and half-transporters were found in insects 2,40 . A human MRP can be classified as a "long" MRP or "short" MRP based on whether it contains a third N-terminal transmembrane domain (TMD0). If it contains a TMD0, it is considered to be a "long" MRP, (HsABCC1-3, HsABCC6, and HsABCC10); on the contrary, the "short" MRPs include HsABCC4, HsABCC5, HsABCC11, and HsABCC12 41 . In insects, it has been reported that ABCC is involved in insecticide resistance; for instance, when the nymphs of Nilaparvata lugens are exposed to triazophos, the transcript level of an ABCC shows a significant increase 42 . In Pediculus humanus, silencing PhABCC4 by RNAi leads to an increased susceptibility to ivermectin 43 .
In D. citri, five ABCC transporter genes were identified; all genes contained full-length ORFs and encoded full ABC transporters (Table 1, Fig. 2). In phylogenetic analysis ( Supplementary Fig. S3), DcABCC1 clustered with Btabq019529.2 and Btabq000311.1, DmCG6214, two B. mori ABCCs, four human MRPs (HsABCC1, HsABCC2, HsABCC3, and HsABCC6), twenty-three T. urticae ABCCs, and T. castaneum TcABCC-9A. As an orthologue to human MRPs, DmCG6214 is an ATP-dependent, vanadate-sensitive organic anion transporter and transports developmentally significant hormones, such as ecdysteroid and juvenile hormone 41 . DcABCC2 cluster with three human MRPs (HsABCC5, HsABCC11 and HsABCC12), where HsABCC5 and HsABCC11 act as nucleoside transporters; however, the function of HsABCC12 is unknown 2,44 . DcABCC3 and DcABCC5 were placed in a large clade containing HsABCC4, a large cluster of T. urticae ABCCs, seven B. mori ABCCs, a cluster of T. castaneum ABCC5s, and ten D. melanogaster ABCCs. HsABCC4 has the ability to transport a wide variety of endogenous and xenobiotic organic anionic compounds out of the cell; these substrates also include molecules involved in cellular signaling 2 . DcABCC4 clustered with human HsABCC10, DmCG7806, BmABC010636, Tetur03g07840, Btabq004618.1, and TcABCC-4A, and this clade showed clear orthologous relationships. HsABCC10 is known as a drug-efflux pump because it is involved in the transport of amphipathic anions, leading to resistance to a variety of anticancer drugs 45 . In the transcriptomes of D. citri, the orthologues of CFTR and SUR are not identified. ABCD subfamily. The ABCD subfamily transporters are half-transporters in animals with one TMD and one NBD and play a role in transporting fatty acids and acyl-CoA in peroxisomes 46 . Two ABCD transporter transcripts were identified in the transcriptomes of D. citri, both of which have full-length ORFs. The same number of ABCDs was also found in the genome of most other insects 47 (Table 2). In the phylogenetic tree ( Supplementary  Fig. S4), D. citri DcABCD1 clustered with B. tabaci Btabq026746.1, T. castaneum TcABCD-6A, D. melanogaster DmCG2316, B. mori BmABC004616, T. urticae tetur05g06640, H. sapiens HsABCD1 and HsABCD2. DcABCD2 is located in the HsABCD3 clade. The phylogenetic analysis reflected clear orthologous relationships with ABCD transporter proteins among these species, suggesting that ABCD transporters are highly conserved in animals.
ABCE and ABCF subfamilies. The ABCE and ABCF subfamilies are quite distinct from other ABC transporters because they only contain two linked NBDs and lack TMDs (Fig. 2). In view of the special structure, ABCE and ABCF proteins are involved in biological processes other than transportation. RNAi against Caenorhabditis elegans ABCE, which is also known as an RNase L inhibitor (RLI) in eukaryotes, resulted in embryonic lethality and slow growth, suggesting that ABCE plays a role in the regulation of translation and transcription 48 . In humans, HsABCE1 has an important role in HIV-1 assembly 49 , and HsABCF1 (ABC50) is associated with promoting translation initiation 50 . In insects, injection of dsRNA specific for T. castaneum TcABCE-3A and TcABCF-2A, led to 100% mortality in the larvae of T. castaneum 29 .
One ABCE and four ABCF transporter genes were identified in the D. citri transcriptomes. Most eukaryotes only have one ABCE and three ABCF genes ( Table 2). In the phylogenetic tree ( Supplementary Fig. S5), DcABCE1 showed the highest homology with BmABC010129 and TcABCE-3A. ABCFs clustered into well-supported separate clades, with DcABCF1 and DcABCF2 located in the HsABCF1 clade, and DcABCF3 and DcABCF4 positioned at the HsABCF2 and HsABCF3 clades separately. Phylogenetic analysis revealed that the ABCE and ABCF subfamilies were highly conserved.
Based on the research of predecessors, ABCG half-transporters were only identified in metazoan species except one ABCG gene in P. xylostella (Px007949) 51 . However, full-transporters are widely present in fungi and plants 52,53 . The half-transporters have a reverse domain structure with an NBD at the N-terminus and a TMD at the C-terminus (NBD-TMD), while a functional transporter must be dimeric 15 . In humans, five ABCG transporter family genes have been identified. Among these HsABCGs, four HsABCGs except HsABCG2 were mainly involved in the transportation of dietary lipids, while HsABCG2 (breast cancer resistance protein, BCRP) has a series of substrates, including anticancer drugs, and acts as an MRP 54 . Among invertebrates, D. melanogaster ABCG members were first characterized, including brown, scarlet, and white genes 55 .
Eighteen ABCG transporter family transcripts were identified in the transcriptomes of D. citri and represent the largest ABC subfamily in D. citri, all of which possess full-length coding sequences and are in accord with half-transporters with the topology TMD-NBD. In the phylogenetic tree (Fig. 3), eight D. citri ABCG genes (DcABCG1-3, DcABCG5-8, and DcABCG18) clustered with potential orthologues of HsABCG1 and HsABCG4 in ABCG clades, where HsABCG1 is involved in regulating the output of cholesterol, while the function of HsABCG4 was not clear 56 . In humans, HsABCG5 and HsABCG8 form a functional heterodimer and play a role in removing plant sterols from the body 56 . In the phylogenetic tree, DcABCG9 and DcABCG10 were two orthologous genes of HsABCG5 and HsABCG8, and all the arthropod orthologues of HsABCG5/HsABCG8 showed a head-to-head arrangement, indicating that DcABCG9 and DcABCG10 may have similar functions as HsABCG5/ HsABCG8. Six genes (DcABCG12-17) clustered with D. melanogaster white, scarlet, and brown and the orthologues of the other species. In D. melanogaster, white, scarlet, and brown are the best-characterized ABCG genes of arthropods, and scarlet or brown takes part in transporting pigment precursors in the Malpighian tubules and relates to the formation of compound eye colour 57,58 . D. melanogaster white mutants show a white-eye colour phenotype, and this phenomenon has also been confirmed in T. castaneum and B. mori 29,59 . However, white is also involved in resistance to pesticide, and downregulation of the white orthologues leads to increased Bt resistance in P. xylostella 60 . In D. citri, DcABCG17 is orthologous to white, DcABCG13 and DcABCG14 are orthologous to scarlet, and three genes (DcABCG12, DcABCG15 and DcABCG16) are orthologous to brown. DcABCG11 clustered with B. tabaci Btabq023890.1, T. castaneum TcABCG-8A, D. melanogaster DmCG3327, and T. urticae tetur17g02510. In D. melanogaster, DmCG3327 (also named E23) is capable of modulating the 20E response 61 , and a similar function has also been found in T. castaneum TcABCG-8A 29 . DcABCG6 clustered with T. castaneum TcABCG-4C, D. melanogaster DmCG3164, B. mori BmABC005202 and two orthologues of B. tabaci. In T. castaneum, TcABCG-4C-dsRNA injected larvae exhibited a rough cuticle as a consequence of desiccation and shrinkage and rapidly caused death during the quiescent stage, in addition, injection of TcABCG-4C-dsRNA into pre-pupae resulted in death at the pupal stage before the pupal-adult molt, while DmCG3164 performs a similar function in Drosophila 29,62 . ABCH subfamily. The ABCH transporter family proteins are half-transporters and share the same NBD-TMD topological structure as the ABCH family. ABCH transporters were first identified in D. melanogaster and it was then reported that the ABCH subfamily was only found in arthropods, zebrafish Danio rerio and marine medaka Oryzias melastigma 15,63 . At present, the ABCH subfamily has not been identified in other species such as mammals, plants and fungi 2,52,64 . We identified fifteen DcABCH genes in the transcriptomes of D. citri, fourteen of them have the full-length ORF. In most insect species, only three ABCH members were found, including D. melanogaster, B. mori and T. castaneum which has an excellent genome sequence, however, a large number of ABCH members were found in three Hemiptera insects (B. tabaci, Lygus Hesperus and D. citri) and two species of arthropods (D. pulex and T. urticae) ( Table 2). In the phylogenetic tree (Fig. 4), six DcABCH sequences (DcABCH1, DcABCH4, DcABCH7, DcABCH12 and DcABCH14-15) formed a separate clade, which was similar to the ABCHs of T. urticae 30 and D. pulex 65 , suggesting that the diversity of ABCH proteins in D. citri  www.nature.com/scientificreports www.nature.com/scientificreports/ has been due to lineage-specific duplications events, this similar expansion was also found in two Hemiptera insects B. tabaci and L. hesperus 26,64 .

Species
ABCH plays an important role in insect physiology. In recent years, researchers have been exploring and have a considerable understanding of their physiological function. In Helicoverpa armigera and Manduca sexta, when the larvae were fed with secondary metabolites, the expression of ABCH subfamily was induced to increase 66,67 . In D. melanogaster, cold hardening treatment resulted in a 2-fold increase in the expression level of DmCG33970 68 , both DmCG9990 null mutants and RNAi-mediated knockdown DmCG9990 are lethal 62,69 . In addition, DmCG9990 was also found to be associated with the formation of epidermal barrier 70 . In T. castaneum, injection of dsRNA specific for TcABCH-9C, the ortholog of DmCG9990, resulted in desiccation and 100% larval mortality and a significant reduction in fertility and the egg hatchability. Furthermore, TcABCH-9C dsRNA treated larvae showed a lack of lipids in the epicuticle, and based on these results, the authors inferred that TcABCH-9C was involved in transport of lipids to epicuticle and promoting the formation of waterproof barrier in epicuticle 29 . In a recent study, the ortholog of DmCG9990 in Locusta migratoria, LmABCH-9C, were borne out to be associated with transport of lipids to epicuticle and cuticle barrier formation in epicuticle 71 . In D. citri, DcABCH5 and DcABCH9 are orthologues of DmCG9990 and DmCG33970, respectively (Fig. 4).
Expression profile of DcABCs. The spatial expression profiles of these ABC transporter genes were estimated by analysing the FPKM values. Ten genes (DcABCB5, DcABCD1, DcABCE1, DcABCF2-3, DcABCG6-8, DcABCG10, and DcABCH5) are widely expressed in adult tissues of D. citri. Seven ABC transporter genes (DcABCA1, DcABCA3-4, DcABCC1, DcABCC3-4, DcABCG15, and DcABCH2) showed high expression levels in the abdomen of adults (Fig. 5). To understand the possible role of DcABCs in the insecticide resistance of D. citri, qRT-PCR was used to compare the expression of these genes between the imidacloprid-susceptible and imidacloprid-resistant strains. The expression level of DcABCG11 in susceptible strains was too low to be detected; therefore the expression levels of 52 genes were presented. Nine DcABCs were significantly upregulated in the imidacloprid-resistant strains compared to the susceptible strains (Fig. 6), and the upregulated genes were mainly concentrated in the ABCG and ABCH subfamilies. DcABCH4 was upregulated 3.9-fold in www.nature.com/scientificreports www.nature.com/scientificreports/ resistant strains, and DcABCG3 and DcABCG9 were upregulated 2.6-fold and 2.9-fold. DcABCG6, DcABCG7, DcABCG10, DcABCH5, DcABCH9, and DcABCH12 were upregulated 1.4-fold to 1.8-fold. Twenty-seven of the 52 DcABCs were down-regulated in resistant strains, and the expression of the remaining 16 DcABCs did not show a significant difference between susceptible and resistant strains.
In arthropods, ABCB, ABCC and ABCG were the three most reported subfamilies associated with insecticide transport or resistance, and the index of insecticide transport or resistance is mainly based on their expression level 15 . In D. citri, five ABCG transporters were significantly upregulated in imidacloprid-resistant strains, and similar upregulation of expression has also been reported in other insects. For instance, one ABCG transporter was upregulated in the imidacloprid-susceptible Leptinotarsa decemlineata 72 . Four ABCG transporters were up-regulated in imidacloprid-treated B. tabaci 64 . In addition to the ABCG transporter, four ABCH transporters were significantly upregulated in imidacloprid-resistant strains. In the green peach aphid, Myzus persicae, an ABCH was upregulated under the stress of pirimicarb 73 , suggesting that ABCH may also be involved in insecticide resistance in insects. The results indicated a potential implication of these genes in imidacloprid resistance. In addition, five of the nine upregulated genes (DcABCG6-8, DcABCG10, and DcABCH5) were expressed widely in adult tissues, which may demonstrate that the wide expression of ABC transporters may contribute to the transport of exogenous substances such as pesticides. Twenty-seven DcABCs were downregulated in resistant strains. Similarly in P. xylostella, Px006766 (PxABCF3) and Px013659 (PxABCA12) were downregulated in chlorpyrifos-resistant and fipronil-resistant strains 51 . This may indicate that not all ABCs are involved in detoxification and may be a physiological adaptation to long-term pesticide pressure.

Conclusions
The major objective of this study was to identify the ATP-binding cassette transporter gene family in D. citri. In this study, fifty-three genes encoding ABC transporters were identified in D. citri using RNA-Seq and transcriptomic analysis. Among 8 subfamilies, ABCG and ABCH have more members in D. citri. Moreover, nine genes of these two subfamilies were upregulated in the imidacloprid-resistant strain of adult D. citri and five of them were expressed extensively in adult tissues. These results enrich the research content regarding the insecticide resistance mechanism in D. citri and will further facilitate our understanding of imidacloprid-resistance mechanisms in this pest. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Insect rearing and strains. Two strains of D. citri were used in this study: laboratory-susceptible strains and imidacloprid-resistant strains. The laboratory susceptible-strains were collected from Murraya exotica on the campus of South China Agricultural University, Guangzhou, Guangdong Province, China, in 2013, and this population was reared in the greenhouse without exposure to any insecticides. The imidacloprid-resistant strains originate from field populations in Guangzhou, Guangdong Province, China, in 2016, and then they were continuously exposed to imidacloprid to select the resistant strains. A 52.19-fold imidacloprid-resistant strain was obtained through 9 generations of continuous selection via the leaf dip method 25 . Both strains were kept rearing on M. exotica in a climate chamber (26 °C, 80% RH) and a 14:10 h (light:dark) photoperiod.
Sample collection and RNA Seq. The tissues of insects were dissected from 3-day-old adults of laboratory-susceptible strains. A total of 2000 antennas (including a modicum of tissues of heads), 200 heads (antennas removed), 150 thoraxes, 300 legs, 150 abdomens, and 1000 terminal abdomens (cut from the 5th abdominal segments) were collected from male adults, and the tissues from famale adults had equal numbers. Total RNA from each sample was extracted using TRIzol Reagent (Invitrogen, Waltham, MA, USA). Total RNA samples were quantified and assessed for quality by a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Transcriptome sequencing was performed on an Illumina HiSeq. 2500 platform (Genewiz, Suzhou, China), and a total of 143.37 Gb of raw data was acquired. After removing low-quality, adaptor and contaminating sequence reads, 137.22 Gb of clean reads was obtained. The clean data were assembled by Trinity, and 297,614 unigenes larger than 200 bp were obtained, the unigenes were submitted to InsectBase (http://www. insect-genome.com/data/Diaphorina_citri.transcript.fa.tar.gz) [74][75][76] . The raw data of the transcriptomes were submitted to the NCBI Short Read Archive (SRA) database (Submission ID: SRP139008) (https://www.ncbi.nlm.nih. gov/sra/SRP139008) 76 .   30 , and Saccharomyces cerevisiae 77 were used as BLAST queries with an E-value threshold of 10 −5 . To obtain the whole ABC transporter genes as far as possible, the same method was also used to identify ABC transporters in the genome of D. citri (Accession: GCF_000475195.1). The candidate ABC transporter genes were reconfirmed by BLASTx analysis with the non-redundant protein sequence (NR) of NCBI (http://www.ncbi.nlm.nih.gov/).

Phylogenetic analysis.
To classify the position of D. citri ABCs within ABC classes (A-G), the amino acid sequences of NBDs of D. citri ABC transporters were used to resolve their phylogenetic relationships. When a protein had two NBDs, the N-terminal NBD was used. To analyse the evolutionary placement of ABC transporters in D. citri, comparison analyses among each subfamily of ABC transporters from D. citri, D. melanogaster, B. mori, T. castaneum, B. tabaci, and T. urticae were conducted, and the full-length protein sequences were subjected to phylogenetic analyses (Supplementary data). Sequences were aligned by the ClustalW alignment algorithm 82 , and MEGA6 was used to construct the neighbor-joining trees with the Poisson model and 1,000 bootstrap replicates 83   www.nature.com/scientificreports www.nature.com/scientificreports/ which is based on the number of uniquely mapped reads 85 . The FPKM values of each gene were transformed into log2 (RPKM + 1) values, and GraphPad Prism 7.01 was used to generate and visualize the expression profile.
The CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) and the Go Taq ® qPCR Master Mix (Promega, Madison, WI, USA) were used to perform qRT-PCR reactions. Finally, the relative values of mRNA expression were calculated by The 2 −ΔΔCt method 88 , and the expression level of imidacloprid-susceptible strain was used as the calibrator. The significance of differences between two strains was determined using Student's t test (P < 0.05). Three biological replicates were analysed for each experiment. A total of 120 ACP adults (three biological replicates, n = 40) from susceptible and resistant strains were used for qRT-PCR analysis, and three technical replicates were performed in each qRT-PCR reaction.