Identification and Expression Characterization of ATP-Binding Cassette (ABC) Transporter Genes in Melon Fly

Simple Summary The melon fly, Zeugodacus cucurbitae, is an important agricultural pest. At present, chemical pesticide treatment is the main method for field control, but this promotes pesticide resistance by Z. cucurbitae, because of its frequent use. ABC transporters are involved in detoxification metabolism, but few studies have yet considered their expression in melon fly. In this study, we identified the ABC transporters genes at a genome-wide level in melon fly, and analysed their spatiotemporal expression patterns, as well as changes in expression after insecticides treatments. A total of 49 ABC transporters were identified, and their expression levels varied at different developmental stages and between tissues. After three insecticides treatment, ZcABCB7 and ZcABCC2 were up-regulated. After β-cypermethrin induction, tissues were dissected at 12, 24 and 48 h, and the expression levels of a number of ABC genes were highly expressed within the fat body. From these results, we conclude that ZcABCB7 and ZcABCC2 may be involved in detoxification metabolism, and that the fat body is the main tissue that plays this role. Abstract The ATP-binding cassette (ABC) transporter is a protein superfamily that transports specific substrate molecules across lipid membranes in all living species. In insects, ABC transporter is one of the major transmembrane protein families involved in the development of xenobiotic resistance. Here, we report 49 ABC transporter genes divided into eight subfamilies (ABCA-ABCH), including seven ABCAs, seven ABCBs, 10 ABCCs, two ABCDs, one ABCE, three ABCFs, 16 ABCGs, and three ABCHs according to phylogenetic analysis in Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. The expressions level of 49 ABC transporters throughout various developmental stages and within different tissues were evaluated by quantitative transcriptomic analysis, and their expressions in response to three different insecticides were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). These ABC transporter genes were widely expressed at developmental stages but most highly expressed in tissues of the midgut, fat body and Malpighian tube. When challenged by exposure to three insecticides, abamectin, β-cypermethrin, and dinotefuran, the expressions of ZcABCB7 and ZcABCC2 were significantly up-regulated. ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were significantly up-regulated in the fat body at 24 h after β-cypermethrin exposure. These data suggest that ZcABCB7 and ZcABCC2 might play key roles in xenobiotic metabolism in Z. cucurbitae. Collectively, these data provide a foundation for further analysis of ABCs in Z. cucurbitae.


Introduction
ATP-binding cassette (ABC) proteins comprise one of the largest superfamilies of prokaryote and eukaryote membrane proteins [1]. According to sequence homology and structural similarity, they can be divided into different subfamily members. For example, a total of 48 ABC transporters in humans are divided into seven subfamilies (ABCA to ABCG) [2], while in Drosophila melanogaster, an eighth subfamily (ABCH) was first discovered. This latter member is widely expressed in arthropods and zebrafish but not in mammals, plants and fungi [3,4]. The majority of these ABC proteins comprise two highly conserved structural domains, a nucleotide-binding domain (NBD) that can bind and hydrolyze ATP, and a transmembrane domain (TMD) that is comprised of six membrane-spanning helices [5]. The highly conservative NBD contains three motifs: a Walker A and Walker B domain and the ABC signature motif (LSSG-motif). There are two of these structural domains in ABC transporters from eukaryotes. These are either half-transporters which have one TMD and one NBD, or full-transporters which have two TMDs and two NBDs, and often form a functional unit as a homo-or heterodimer [1,6]. Up until now, the ABC transporters have been widely identified at a genome-wide level in many insect species.
In terms of different functions, ABC transporters can be divided into three categories: input proteins, output proteins and non-transport proteins [7]. The main function of the input and output proteins is substrate transport, e.g., amino acids, lipids, lipopolysaccharides, peptides, inorganic ions, drugs, metals and xenobiotics [8]. Non-transport proteins have no transport function, but are involved in intracellular DNA repair, transcription and regulation of gene expression [9]. In insects, studies have shown that ABC transporters have roles in development and detoxification, including the metabolism of pesticides, plant secondary metabolic compounds (allelochemicals) and other heterologous substances [10]. In recent years, the important role of ABC transporters in the insect's response to pesticides and other toxic compounds has caused widespread concern [11,12]. In Bemisia tabaci, the ABCG3 subfamily may contribute to imidacloprid detoxification [13]. In Plutella xylostella, four ABC subfamilies (ABCA, ABCC, ABCG, and ABCH) were increased in a fipronil resistant strain [14]. Similarly, after insecticide exposure, some ABC transporters were up-regulated in mosquito larvae [15]. In Aphis gossypii, ABC transporters are involved in thiamethoxam resistance [16]. In addition, inhibition of ABC transporter gene expression by RNA interference (RNAi) can increase pesticide toxicity [17,18].
Although the contribution of ABC transporters to drug resistance in mammals has been extensively studied, the mechanisms by which these ABC transporter genes confer resistance in arthropods remains unclear. The ABCB subfamily contains multi-drug resistance proteins (MDRs) or P-glycoproteins (P-gps). The ABCC subfamily includes the multidrug-resistance associated proteins (MRPs) in humans, and these are involved in the multidrug resistance of cancer cells to chemotherapy [4]. The detoxification processes in insects are divided into three phases: cytochromes P450 monooxygenase (P450s) and carboxylesterases (CarEs) belong to phase I metabolism, glutathione S-transferases (GSTs) belongs to phase II, while ABC transporters belongs to phase III. Phases I and II can act directly upon a toxin molecule, and then the substrates transported extracellular in phase III [19,20]. Therefore, it is crucial to understand the role of ABC for conferring resistance to insecticides.
The melon fly Zeugodacus cucurbitae (Coquillett) (Diptera, Tephritidae) is an important, global, agricultural pest [21]. Z. cucurbitae can infest more than 130 host plants including vegetables and fruits, but mainly gourd and nightshade plants, such as cucumber, pumpkin, melon, watermelon, bitter melon, tomato and eggplant [22] Although the biological control by releasing of parasitoids against Z. cucurbitae was evaluated recently [23], the extensive use of pesticides is the frequently method to control Z. cucurbitae, which leads to serious resistance to a range of insecticides. At present, there are few studies that have considered the ABC transporters present within the melon fly. Moreover, whether ABC proteins are also involved in different insecticide detoxifications has not been investigated in Z.
cucurbitae. In this study, we identified 49 ABC transporters from Z. cucurbitae. They were divided into eight subfamilies of ABCA~ABCH. The gene expression profiles were studied at different development stages and within various tissues. Secondly, we focused on an analysis of the expression of ABC transporters after three different insecticide treatments. Collectively, a more comprehensive analysis of ABC genes will provide an insight into the physiological function of ABC transporters, and how this is regulated in response to xenobiotic stimuli.

Insects
Z. cucurbitae were initially collected from Haikou of Hainan Province, China, in 2016, and maintained in the laboratory under conditions of 27 ± 0.5 • C, 14-h L: 10-h D photoperiod, 70 ± 5% relative humidity (RH). The insects were fed on an artificial diet, as previously described [24].

Identification, Characterization and Phylogenetic Analysis
To identify ABC transporter genes, we carried out tBlastn searching against the Z. cucurbitae genome [25], with highly conserved NBDs present in D. melanogaster ABC transporter genes. The putative ABC gene sequences were taken as the query objects, and each of the ABC transporter genes was confirmed in the NCBI protein database.

RNA Isolation and cDNA Synthesis
Three tissues (midgut, fat body, and Malpighian tubule) were dissected from 20 female adult flies, with four replicates of each that includes 4-5 individuals. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA according to the manufacturer's instructions. The concentration and purity of 1.5 µL of dissolved RNA for each of the samples were quantified using a Nanodrop ONE spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at absorbance ratios of OD 260/280 and OD 260/230. The integrity of RNA was further confirmed after resolution by 1.0% agarose gel electrophoresis. Synthesis of cDNA was performed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) to remove genomic DNA, following the manufacturer's protocol.

Expression Profiles
Expression profiling of ABC transporters was evaluated using transcriptome data. To determine the expression patterns of ABC transporter genes, the midgut, fat body, Malpighian tubule, ovary and testis of male and female, respectively, were harvested at the following developmental stages: egg; 1-day, 3-day, and 7-day-old larva; 1-day, 5-day, and 9-day-old pupa, as well as 1-day, 5-day, and 9-day-old male or female adults. Total RNA and cDNA preparation was performed as described above. The real-time quantitative PCR (RT-qPCR) was run on a CFX384 Real-time System (Bio-Rad, Singapore) using the NovoStar SYBR qPCR SuperMix (Novoprotein Scientific, Shanghai, China). The qRT-PCR program was run with the following conditions: 95 • C for 2 min, 40 cycles of 95 • C for 15 s, 60 • C for 30 s, and 72 • C for 30 s. A melting curve analysis at 60-95 • C was performed to ensure the specificity of each primer. In addition, α-tubulin and β-tubulin 1 [29] were used as reference genes for normalization. The primers used for ABC transporters validation were designed using Primer 3.0 (http://bioinfo.ut.ee/primer3-0.4.0/) (Table S1). All reactions were undertaken with two technical replicates and three biological replicates. The relative expression level of the ABC genes was calculated using qBase [30].

Insecticide Exposures
The transcriptional responses of certain ABC genes of Z. cucurbitae to three commonly used insecticides were investigated, including one neonicotinoid, dinotefuran (98% purity, Hailir, Shandong, China), one biogenic pesticide, abamectin (95% purity, Bangnong, Guangdong, China), and one pyrethroid, β-cypermethrin (93% purity, LIER-Chemical, Sichuan, China). Insecticides were dissolved in acetone, and then diluted to achieve five or six different concentrations: dinotefuran (at 1000, 700, 500, 250, 100 mg/L), abamectin (at 120, 60, 30, 15, 7.5, 3.75 mg/L) and β-cypermethrin (at 480, 240, 120, 80, 60 mg/L). Newly emerged adults were collected and sexually separated in two cages. Five-day-old virgin female adults were anesthetized by exposure to CO 2 for 2 min and then treated with insecticide (0.5 µL per fly) by dropping it onto their pronotum. For each concentration, approximately 20 adult flies were placed in a plastic cup. Z. cucurbitae were treated with acetone solutions as control. Each concentration of the insecticides was performed with three replicates. After 48 h, the mortality of Z. cucurbitae was recorded. If the tested flies did not move after stimulation with a camel hairbrush, they were considered to be dead. The concentration producing either 50% or 30% lethality (LC 50 and LC 30 , respectively) was calculated using PoloPlus. Three insecticides, dinotefuran (LC 50 , 296.01 mg/L; LC 30 , 219.62 mg/L), abamectin (LC 50 , 14.66 mg/L; LC 30 , 9.34 mg/L) and β-cypermethrin (LC 50 , 125.91 mg/L; LC 30 , 100.58 mg/L) were used in this study. Insecticides were diluted in acetone at the experimentally-determined LC 30 and LC 50 concentrations. Using the same method (as above) to treat five-day-old virgin female adults, acetone was used as control. In total, 120 adults were treated with insecticides. Surviving flies after each treatment were randomly collected for RNA extraction at 12 h, 24 h and 48 h post-exposure. Four adults from each time points were collected and RNA extracted. There were four biological replicates for each treatment. Ribosomal protein subunit 3 and Ribosomal protein L13 were selected as reference genes evaluated before the quantification (Table S1).

Statistical Analysis
Relative gene expressions within tissues at each developmental stage were analyzed using SPSS 25.0 (SPSS Inc., Chicago, IL, USA). A p < 0.05 was regarded as statistically significant.

Identification and Phylogenetic Analysis of ABC Transporter Genes
Based upon the genome sequence, we identified 49 putative ABC genes in Z. cucurbitae. These genes were divided into eight subfamilies: seven ABCAs, seven ABCBs, 10 ABCCs, two ABCDs, one ABCEs, three ABCFs, 16 ABCGs, and three ABCHs (Table 1), according to the highly conserved NBDs of D. melanogaster ABC transporter genes. All of the putative genes had complete ORFs. The 49 ABC transporters were retrieved using the NCBI protein database with its known homologous sequences. The NBD and TMD were further determined using the Pfam program to determine the 49 ABC transporters. Each potential ZcABCs is classified into two possible structural forms, full transporters (FTs) or half transporters (HFs). All of the ABCA subfamily belongs to FTs, ZcABCD, ZcABCG, and ZcABCH belong to HFs, and ZcABCG13 belongs to the FTs family. While some of the genes such as ZcABCB and ZcACC belong to FTs and the others belong to HFs. ZcABCA, ZcABCB, ZcABCC and ZcABCD subfamilies contained both TMD and NBD organization, but ZcABCG and ZcABCH subfamilies formed NBD-TMD organization. However, no TMD motif was predicted for the ZcABCEs and ZcABCFs subfamilies.

Subfamily Gene Gene IDc Length (AA) Topology a N-Glc b O-Glc c
a Transmembrane helices (TMs) were predicted using the TMHMM 2.0 server. b A N-glycosylation sites were predicted using NetNGlyc 1.0 server; only N-glycosylation site with a "potential" score > 0.5 and with a jury agreement were considered. c O-glycosylation sites were predicted using NetOGlyc 4.0 server. If the G-score was higher than 0.5 the residue was considered to be O-glycosylated, the total number of O-glycosylated sites (glycosylated serine and threonine) is shown.
To determine the homology and phylogenetic relationships, 49 ABC transporter proteins were derived from the NCBI Nt database. A phylogenetic tree was constructed based upon presence of ZcABC transporter proteins and DmABC transporter proteins using NDBs domains. Phylogenetic analysis indicated that melon fly ABC transporters can be divided into eight subfamilies (listed A-H) ( Figure 1). Phylogenetic analysis revealed that ZcABCs were highly similar to DmABCs, with the same subfamily clustered on the same branches.
sidered. c O-glycosylation sites were predicted using NetOGlyc 4.0 server. If the G-score was higher than 0.5 the residue was considered to be O-glycosylated, the total number of O-glycosylated sites (glycosylated serine and threonine) is shown.
To determine the homology and phylogenetic relationships, 49 ABC transporter proteins were derived from the NCBI Nt database. A phylogenetic tree was constructed based upon presence of ZcABC transporter proteins and DmABC transporter proteins using NDBs domains. Phylogenetic analysis indicated that melon fly ABC transporters can be divided into eight subfamilies (listed A-H) ( Figure 1). Phylogenetic analysis revealed that ZcABCs were highly similar to DmABCs, with the same subfamily clustered on the same branches.

The Expression Pattern of ZcABCs
The expressions of 49 ABC transporter genes across developmental stages and tissues in Z. cucurbitae adults with RPKM > 0 were analyzed. Regarding developmental stages, the majority of ZcABCs were expressed at low levels in egg and larva stages. We detected multiple Z. cucurbitae ABC genes that are highly expressed during the pupation and adult stage. The analysis revealed that the expression of eight ABC genes and nine ABC genes were expressed in increasing amounts as the female adult and the male adult developed, respectively. Some ABC genes are highly expressed in one day pupa ( Figure 2). in Z. cucurbitae adults with RPKM > 0 were analyzed. Regarding developmental stages the majority of ZcABCs were expressed at low levels in egg and larva stages. We detected multiple Z. cucurbitae ABC genes that are highly expressed during the pupation and adul stage. The analysis revealed that the expression of eight ABC genes and nine ABC genes were expressed in increasing amounts as the female adult and the male adult developed respectively. Some ABC genes are highly expressed in one day pupa (Figure 2). Letters on the right are the gene names. The mRNA levels, represented by normalized log2 (FPKM + 1) values, are shown in the gradient heat map with colors ranging from blue (low expression) to red (high expression). L1, 1-day-old larva, L3, 3-day-old larva, L7, 7-day-old larva. In the same way, P represent pupa, F represent female adult and M represent male adult, the numbe represents the day.
Moreover, we evaluated the levels of 49 ZcABCs transporters genes in different tis sues. More than half of the Z. cucurbitae ABC genes were expressed in Malpighian tubule and midgut (Figure 3). Some tissue-specific ABC genes were identified, e.g., ZcABCA2 ZcABCA3, ZcABCA4, and ZcABCA5 in the testis, and ZcABCA6, ZcABCB4, ZcABCE1 ZcABCF2 in the ovary. Some of these genes corresponded to the high expression at adul stage. Letters on the right are the gene names. The mRNA levels, represented by normalized log 2 (FPKM + 1) values, are shown in the gradient heat map with colors ranging from blue (low expression) to red (high expression). L1, 1-day-old larva, L3, 3-day-old larva, L7, 7-day-old larva. In the same way, P represents pupa, F represents female adult and M represents male adult, the number represents the day.
Moreover, we evaluated the levels of 49 ZcABCs transporters genes in different tissues. More than half of the Z. cucurbitae ABC genes were expressed in Malpighian tubule and midgut (Figure 3). Some tissue-specific ABC genes were identified, e.g., ZcABCA2, ZcABCA3, ZcABCA4, and ZcABCA5 in the testis, and ZcABCA6, ZcABCB4, ZcABCE1, ZcABCF2 in the ovary. Some of these genes corresponded to the high expression at adult stage.

Transcriptional Responses of ZcABCs to Insecticides Exposures
In order to evaluate the relative expression of ZcABCBs and ZcABCCs in response to insecticides exposures, five-day-old female adults were firstly treated with different insecticides and the levels of mortality quantified. The LC 50 values of the insecticides for dinotefuran, abamectin and β-cypermethrin were 296.010 mg/L, 14.658 mg/L, and 125.908 mg/L, respectively ( Table 2). The 95% confidence intervals of the LC 30 and LC 50 values for these insecticides were also calculated.
The dosage of LC 30 and LC 50 insecticides were thereafter used to evaluate the induction of gene expression of ABC genes. The results showed that only ZcABCB7 and ZcABCC2, among 17 selected genes that are mainly involved in detoxification metabolism, were remarkably over-expressed in Z. cucurbitae when challenged by a LC 30 of these three  (Figure 4). For β-cypermethrin, ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were significantly up-regulated 24 h after a LC 30 dosage. ZcABCB2, ZcABCC1, and ZcABCC6 were significantly down-regulated by the LC 30 dosage, and ZcABCB3 and ZcABCB4 significantly down-regulated after 48 h ( Figure 4A). After exposure to abamectin, ZcABCB2, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, ZcABCC7, and ZcABCC9 were significantly up-regulated 12 h after dosage with the LC 30 . The mRNA expression levels were up-regulated from 1-to 2.5fold. ZcABCB1, ZcABCB4, ZcABCB6, ZcABCC1, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC6 were all significantly down-regulated 24 h and 48 h after a LC 50 dosage ( Figure 4B). After a challenge with dinotefuran, only the expression of ZcABCB7 was significantly up-regulated 12 h after a LC 30 dosage. ZcABCC1, ZcABCC2, and ZcABCC10 were significantly up-regulated 48 h after a LC 30 and 24 h after LC 50 treatments ( Figure 4C).

Transcriptional rResponses of ZcABCs to Insecticides Exposures
In order to evaluate the relative expression of ZcABCBs and ZcABCCs in response to insecticides exposures, five-day-old female adults were firstly treated with different insecticides and the levels of mortality quantified. The LC50 values of the insecticides for dinotefuran, abamectin and β-cypermethrin were 296.010 mg/L, 14.658 mg/L, and 125.908 mg/L, respectively ( Table 2). The 95% confidence intervals of the LC30 and LC50 values for these insecticides were also calculated.
The dosage of LC30 and LC50 insecticides were thereafter used to evaluate the induction of gene expression of ABC genes. The results showed that only ZcABCB7 and

Transcriptional Response of ZcABCs to β-Cypermethrin Exposure in Tissues
Expression levels of ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 within several tissues important for detoxification (midgut, fat body, and Malpighian tubules) were quantified at 12 h and 24 h after a LC30 exposure to β-cypermethrin. All of these ABC transporter genes were significantly induced within the fat body 24 h after exposure ( Figure 5), especially ZcABCB7 and ZcABCC2 with a 4.25 and 3.30-fold change, respectively ( Figure 5C,D). In addition, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, and ZcABCC7 were significantly down-regulated in Malpighian tubules 12 h after exposure. The 17 genes showed no significant expression changes within the midgut,

Transcriptional Response of ZcABCs to β-Cypermethrin Exposure in Tissues
Expression levels of ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 within several tissues important for detoxification (midgut, fat body, and Malpighian tubules) were quantified at 12 h and 24 h after a LC 30 exposure to β-cypermethrin. All of these ABC transporter genes were significantly induced within the fat body 24 h after exposure ( Figure 5), especially ZcABCB7 and ZcABCC2 with a 4.25 and 3.30-fold change, respectively ( Figure 5C,D). In addition, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, and ZcABCC7 were significantly down-regulated in Malpighian tubules 12 h after exposure. The 17 genes showed no significant expression changes within the midgut, although ZcABCB7, ZcABCC3, ZcABCC4, and ZcABCC7 were down-regulated markedly after both 12 h and 24 h.

Discussion
With the relatively recent improvements in sequencing technology, more genomes of arthropods have been sequenced and annotated, leading to an expansion of databases and ability to identify family genes. For example, there are 56 ABC transporter genes in D. melanogaster [31], 55 in B. tabaci [32], 55 in Bombyx mori, 73 in Tribolium castaneum, 41 in Apis mellifera, 52 in Anopheles gambiae [33], and 44 in Diaphorina citri [34]. In this study, a total of 49 ABC transporter genes were identified in Z. cucurbitae based upon the sequenced genome. These genes were divided into eight subfamilies (ABCA-ABCH) which varied based upon their functions and similarity of conserved domains. Compared to D. melanogaster, the A and C subfamily of Z. cucurbitae have relatively small numbers. The Z. cucurbitae ABCA subfamily has six FT and one incomplete ABC protein with one NBD domain. In silkworm, ABCAs consist of two FT, one HT, and only three have one NBD [35]. In Bactrocera dorsalis, ABCAs consist of five FT and two HT [36]. In contrast, all of the ABCAs subfamily genes were FTs in A. gambia [12].
The structural differences between the ABCA subfamily indicate diversity associated with the evolutionary process in insects. The analysis of the ABCB subfamily of Z cucurbitae revealed three FT and four HT members. This differs from that of B. tabaci in which all ABCBs members are HTs. Both FTs and HTs play important roles in arthropods [37]. For the ABCD, ABCE, ABCF, and ABCH subfamilies of Z cucurbitae, two, one, three, and three proteins were identified, respectively. The same numbers of these subfamily genes were also identified in other insects, indicative of a conserved evolution.
The ABCG genes constitute the largest subfamily, and the ABCH subfamily is similar to ABCG. ABCGs are involved in pigment precursor transporters, such as brown, scarlet and white genes in D. melanogaster [31,38]. The ABCH subfamily was first reported in D. Student's t-tests were performed for comparison with the corresponding control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Discussion
With the relatively recent improvements in sequencing technology, more genomes of arthropods have been sequenced and annotated, leading to an expansion of databases and ability to identify family genes. For example, there are 56 ABC transporter genes in D. melanogaster [31], 55 in B. tabaci [32], 55 in Bombyx mori, 73 in Tribolium castaneum, 41 in Apis mellifera, 52 in Anopheles gambiae [33], and 44 in Diaphorina citri [34]. In this study, a total of 49 ABC transporter genes were identified in Z. cucurbitae based upon the sequenced genome. These genes were divided into eight subfamilies (ABCA-ABCH) which varied based upon their functions and similarity of conserved domains. Compared to D. melanogaster, the A and C subfamily of Z. cucurbitae have relatively small numbers. The Z. cucurbitae ABCA subfamily has six FT and one incomplete ABC protein with one NBD domain. In silkworm, ABCAs consist of two FT, one HT, and only three have one NBD [35]. In Bactrocera dorsalis, ABCAs consist of five FT and two HT [36]. In contrast, all of the ABCAs subfamily genes were FTs in A. gambia [12].
The structural differences between the ABCA subfamily indicate diversity associated with the evolutionary process in insects. The analysis of the ABCB subfamily of Z cucurbitae revealed three FT and four HT members. This differs from that of B. tabaci in which all ABCBs members are HTs. Both FTs and HTs play important roles in arthropods [37]. For the ABCD, ABCE, ABCF, and ABCH subfamilies of Z cucurbitae, two, one, three, and three proteins were identified, respectively. The same numbers of these subfamily genes were also identified in other insects, indicative of a conserved evolution.
The ABCG genes constitute the largest subfamily, and the ABCH subfamily is similar to ABCG. ABCGs are involved in pigment precursor transporters, such as brown, scarlet and white genes in D. melanogaster [31,38]. The ABCH subfamily was first reported in D. melanogaster but has not been found in plant and fungi [32]. Hence, these genes may play unique roles in insects. In Locusta migratoria an ABCH subfamily member LmABCH-9c was demonstrated to be involved in the process of constructing a lipid-based barrier on the cuticle surface [39].
A comprehensive expression pattern of ZcABC genes could provide an insight into potential physiological functions associated with development and/or within tissues. In Z. cucurbitae, ZcABCs were expressed in the larva stage at a relatively low level but with a high expression in egg, pupa, and adult stages. This indicated that most of ZcABCs are involved in metamorphosis development. This is supported by a study of a knockdown of the TcABCA-9A/9B in T. castaneum, as this caused wing defects and elytral shortening at pupa and adult, and knockdown of TcABCE-3A and TcABCF-2A was lethal because of failed molting [40]. In Z cucurbitae, five ZcABCA genes exhibited a higher expression at male adult stage that increased gradually, which is consistent with results in Helicoverpa armigera [41].
Expression of ZcABCBs and ZcABCCs family genes were dynamic at all developmental stages of Z cucurbitae. The expression patterns were different among these genes, indicating that ABCB and ABCC subfamilies probably have minor roles during insect metamorphosis. ABCB and ABCC subfamilies are also thought to be involved in pesticide resistance and other chemicals in insects. ZcABCE1, ZcABCF2, and ZcABCF3 were highly expressed at each stage. Hence, the expression patterns of these three genes are consistent with basic functions of biogenesis and translation regulation of ribosomes [42]. ZcABCGs and ZcABCHs were expressed in a complex fashion at all developmental stages, because of their functional diversity.
The expressions of ZcABCs were diverse within the different tissues. Multiple ZcABCBs and ZcABCCs were highly expressed in the Malpighian tubule and midgut, such as ZcABCC (1)(2)(3)(4)(5)(6)8), ZcABCB (2, 3, 5, 7), and ZcABCG (3,5,9,11,16). Furthermore, the midgut and the Malpighian tubule are a digestive organ and excretory organ, respectively, and are involved in detoxification metabolism. The expression patterns of these genes are similar to most of those from metabolic detoxification families, such as P450, glutathione S-transferase, and carboxylesterase [1]. For example, a knockout of three ABCC subfamily genes (Mdr49, Mdr50, and Mdr65) in D. melanogaster [43], and Mdr49, Mdr50, and Mdr65 resulted in an increased susceptibility to insecticide. In P. xylostella, it has been demonstrated that the pxABCC2 and pxABCC3 proteins also functioned as receptors for BtCry1 toxins within the midgut [44]. These comparable studies provide insights into the potential function of these highly expressed ABC genes within the midgut and Malpighian tubule. The expression of the ZcABC gene within the fat body suggested its potential function in the transport of cuticular lipids. In T. castaneum, an ABC transporter gene plays an important role in cuticle lipid transport to maintain osmotic balance [40]. In this study, some ZcABCs were highly expressed within reproductive tissues. Furthermore, these genes were also expressed in increasing levels as the adult developed. The presence and expression of these genes within testis or ovary indicates that they could play a role in spermatic or ovarian development, respectively.
In addition to the transport of insecticides and allelochemicals, ABC transporters have also been studied as insecticide targets. ABC transporters were involved in the degradation of plant secondary metabolites in the cotton bollworm, H. armigera [5]. ABCBs are often positively correlated with field population resistance and ABCB FTs have been associated with insecticide transport and/or resistance in insects [4]. The ABCC subfamily known as MRPs, transports a range of substrates, such as drugs, endogenous compounds and glutathione, and cyclic nucleotides [16]. In Anopheles arabiensis and D. melanogaster, ABCB and ABCC genes were overexpressed in a dichlorodiphenyltrichloroethane (DDT)-resistant strain [45,46]. Hence, B and C subfamilies were selected for further analysis of the transcriptional response to the insecticides (dinotefuran, abamectin and β-cypermethrin) in this study. An analysis of the expression of the 17 candidate ABC genes was undertaken after insecticide exposure. The results showed that some of ZcABCBs and ZcABCCs were highly expressed after treatment with the insecticides. This positive correlation between induction of the ABC subfamily and insecticide resistance has also been demonstrated in other insects [16]. After insecticide treatment, the ABC gene showed up-regulation in several insects [1]. For example, a CRISPR-Cas9 knockout for Mdr65 was more susceptible to all neuroactive insecticides tested [43]. When imidacloprid was used to treat B. tabaci adults, the expression of ABCG3 was increased, and a knockdown of ABCG3 significantly increased the mortality of adults exposed to imidacloprid [13]. In B. dorsalis, after insecticides exposure, more than 10 ABC genes were significantly overexpressed [36]. Two ABC transporters, ZcABCB7 and ZcABCC2, were significantly up-regulated in Z. cucurbitae after exposure to a LC 30 of three insecticides.
To assess xenobiotic responses, three insecticides were selected, namely, abamectin, βcypermethrin and dinotefuran. We found that most of the ZcABCB and ZcABCC subfamily genes showed no response to dinotefuran. This may result from that neonicotinoid insecticides are more effective to Hemiptera [16,34,47]. However, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were all significantly up-regulated by abamectin andβcypermethrin exposure. Similar results were found in B. dorsalis [36]. A knockdown of BdABCB7 significantly increased the toxicity of malathion, and increased the mortality of B. dorsalis [36]. After exposure to β-cypermethrin, many ABCBs and ABCCs were up-regulated at 24 h, a time point consistent with in other literatures. However, after exposure to abamectin, most of these genes were up-regulated at 12 h, rather than 24 h. Furthermore, the majority of ABCBs and ABCCs had no response to dinotefuran exposure. Therefore, we used β-cypermethrin to test which tissues might play a role in detoxification metabolism After treatment of Z cucurbitae adults with a LC 30 of β-cypermethrin, the midgut, Malpighian tubule, and fat body were dissected 12 h and 24 h after insecticide exposure. The genes: ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were specifically induced within the fat body 24 h after exposure. This result is consistent with the expression pattern of adults after β-cypermethrin exposure, indicating a detoxification related role of these genes in Z. cucurbitae. However, further research is required to fully elucidate this response and its mechanism of action.
Among the seven ABCB transporters identified in Z. cucurbitae, three of them were FTs, and could be participating in detoxification metabolism. In D. melanogaster, three genes (mdr49, mdr50, and mdr65) are part of the ABCB transporters, and disruption of the mdr49 can increase resistance to colchicine [48]. Furthermore, knockout of these three genes renders flies more susceptible to nitenpyram and imidacloprid [43]. A number of invesigations have suggested that these gene products are involved in the transport of these chemicals [49]. In this study, ZcABCB7, which is homologous with mdr49, was mainly expressed in the midgut, fat body and Malpighian tube. The upregulation of ZcABCB7 in Z. cucurbitae by three insecticides exposure indicated an insecticide metabolism related role in Z. cucurbitae.
In addition to the participation of ABCB transporters in insecticide detoxification, ABCC transporters were also found to be involved in insecticide metabolism. A total of 10 ZcABCCs were identified in Z. cucurbitae. They are FTs and may have various functions in ion transport, cell-surface receptor activity and substrate translocation [50]. In humans, the ABCCs mainly comprise MRPs [3]. In Manduca sexta, ABCC transporters are involved in multidrug resistance and detoxification [51]. In Chilo suppressalis, CsABCC8 was significantly up-regulated after chlorantraniliprole exposure and CsABCC8 was homologous with the sulfonylurea receptor (Sur) that may participate in insecticide metabolism [52]. In this study, ZcABCC2 and ZcABCC3 were mainly expressed in adults and within the Malpighian tube. The significant up-regulation of these genes after exposure to three insecticides indicate that they may be involved in insecticide metabolism in Z. cucurbitae.

Conclusions
In summary, herein, a total of 49 ABC transporter genes were identified from Z. cucurbitae. All of these genes had complete ORFs and fitted to a genome scaffold. The phylogenetic relation and subfamily classifications were determined based on the presence of clear homologous orthologs in D. melanogaster. A bioassay that assessed responses to three kinds of insecticides was performed in Z. cucurbitae, and the LC 50 dosage of β-cypermethrin, abamectin and dinotefuran quantified as 125.908, 14.658, and 296.010 mg/mL, respectively. Two prominent subfamily members, ABCB and ABCC, were selected for the evaluation of these three insecticides, with results that indicated that ZcABCB7 and ZcABCC2 may be associated with insecticide tolerance. Dissected tissues from flies exposed to β-cypermethrin, revealed that the ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 genes were significantly expressed in the fat body, implying that the fat body may be the major tissue to transport pyrethroids.