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Transcriptomic insights on the ABC transporter gene family in the salmon louse Caligus rogercresseyi

Parasites & Vectors volume 8, Article number: 209 (2015) Cite this article

Abstract

Background

ATP-binding cassette (ABC) protein family encode for membrane proteins involved in the transport of various biomolecules through the cellular membrane. These proteins have been identified in all taxa and present important physiological functions, including the process of insecticide detoxification in arthropods. For that reason the ectoparasite Caligus rogercresseyi represents a model species for understanding the molecular underpinnings involved in insecticide drug resistance.

Methods

llumina sequencing was performed using sea lice exposed to 2 and 3 ppb of deltamethrin and azamethiphos. Contigs obtained from de novo assembly were annotated by Blastx. RNA-Seq analysis was performed and validated by qPCR analysis.

Results

From the transcriptome database of C. rogercresseyi, 57 putative members of ABC protein sequences were identified and phylogenetically classified into the eight subfamilies described for ABC transporters in arthropods. Transcriptomic profiles for ABC proteins subfamilies were evaluated throughout C. rogercresseyi development. Moreover, RNA-Seq analysis was performed for adult male and female salmon lice exposed to the delousing drugs azamethiphos and deltamethrin. High transcript levels of the ABCB and ABCC subfamilies were evidenced. Furthermore, SNPs mining was carried out for the ABC proteins sequences, revealing pivotal genomic information.

Conclusions

The present study gives a comprehensive transcriptome analysis of ABC proteins from C. rogercresseyi, providing relevant information about transporter roles during ontogeny and in relation to delousing drug responses in salmon lice. This genomic information represents a valuable tool for pest management in the Chilean salmon aquaculture industry.

Background

Caligus rogercresseyi, a sea louse, is a widely prevalent parasite in the Chilean aquaculture industry [1,2]. This ectoparasite belongs to the Caligidae family, which includes species such as Lernaeocera branchialis, Caligus clemensi, and Lepeophtheirus salmonis, with this final species having greater prevalence in countries such as Scotland, Norway, Canada, and England [3]. Although not associated with host mortality, an infection of C. rogercresseyi is a highly stressful condition for fish, which is reflected by lower culture performance and a depression of the host’s immune system, which thus increases susceptibility to other types of contagious diseases [1-4].

Numerous delousing drugs have been used worldwide for the control of this ectoparasite [5]. However, many studies suggest that the effectiveness of different treatments for sea lice principally depends on the developmental stage of the parasite and the delousing drugs used, for example, there are reports that have mentioned the effect of emamectin benzoate in chitin synthesis which results in a fragile exoskeleton after moulting [5-9]. One of the protein families which has shown detoxifying effects against drugs are the ABC transporters. These types of transporters have been widely related to the generation of resistance to emamectin benzoate in L. salmonis [10,11].

The ATP-binding cassette (ABC) proteins family is ubiquitous in the animal kingdom, and has even been found in plants. Most of these ABC proteins are integral membrane proteins that use ATP to transport biomolecules through the plasma membrane [12]. In eukaryotes, this family has a characteristic organization marked by two transmembrane domains (TMD) that are formed by five or six helixes which determine the specificity of the transporter [13,14]. Moreover, this family also presents two cytosolic nucleotide binding domains (NBD), and these bind and hydrolyze the ATP necessary for transporting substances across the membrane [13,14]. The NBD is a highly conserved sequence that presents characteristic sites such as Q-look, the H-motif, and the LSGGQ-motif [14,15]. It is possible to divide ABC transporters into the following two groups: those present only in prokaryotes and which require substrate-binding proteins for transport, and those found only in eukaryotes and that bind directly to the substrate from the interior of the cell [15]. The substances transported by ABC transporters include amino acids, sugars, lipids, inorganic ions, polysaccharides, metals, peptides, and toxic substances [16].

The ABC proteins family is comprised of subfamilies that are differentiated according to domain and sequence structures [16]. In mammals, seven subfamilies (A-G) have been identified, whereas in arthropods and zebra fish, eight subfamilies (A-H) have been found [12,16]. While in prokaryotic organisms such as Escherichia coli, the ABC family has been subdivided into 22 subfamilies with transporter activity and 24 with exporter activity [17]. Of the eight subfamilies describe in arthropod, the E and F subfamilies are the only ones without transporter functions. The ABC transporter E subfamily (ABCE) members act as inhibitors of RNase L and participate in assembling the preinitiation complex, while the ABCF subfamily plays a role in assembling ribosomes and in protein translation [12]. Currently, the ABC transporter subfamilies have only been characterized in eight arthropod species, which are D. melanogaster [13], Anopheles gambiae [18], Apis mellifera [19], Bombix mori [20], Tribolium casteneum [21], Tetranychus urticae [22], Daphia pulex [12], and, recently, Tigriopus japonicus [23].

The ABCB subfamily is especially of interest given its ability to transport drugs [24,25], with P-glycoprotein (P-gp) being the first transporter identified within this family [26]. Furthermore, the ABCC and ABCG families have been reported to have a similar detoxifying function [16,27]. Given their roles related to the detoxification of drugs, these proteins have been termed multidrug resistance proteins (MRPs). In invertebrates, MRPs have been associated with the generation of resistance to insecticides, including in species such as Caenorhabditis elegans, Tricho plusiani [28], and Aedes aegypti [29], among others. In the ectoparasites Lepeophtheirus salmonis [10] and Caligus rogercresseyi [30], a close association has been found between the generation of resistance to emamectin benzoate (EMB) and the transcriptomic response of P-gp. Likewise, an exhaustive study on the different ABC transporter families in D. pulex was carried out with the purpose of understanding the adaptation mechanisms that this crustacean uses in response to toxic compounds [12], and a recent study in L. salmonis characterized an additional four MRPs [31]. However, these MRPs in L. salmonis did not present differences in transcript expression between EMB resistant/susceptible strains.

C. rogercresseyi is an ectoparasite responsible for significant economic losses in the Chilean salmon aquaculture industry, and, as with L. salmonis, this species has demonstrated resistance to the drugs currently used in infestation control [32-34]. Given the role that ABC transporters play in pharmaceutical resistance in invertebrates, the objective of the present study was to identify members of the distinct ABC subfamilies and determine expression patterns during the distinct stages of development in C. rogercresseyi. Moreover, RNA-Seq analysis was performed in adult salmon lice exposed to the delousing drugs deltamethrin and azamethiphos in order to determine the modulation of distinct ABC proteins in response to drugs currently used in the control of C. rogercresseyi.

Methods

Samples and bioassays

Adult male and female sea lice were collected from a commercial farm located in Region de los Lagos of Chile (41°40′48.5″S; 73°02′31.34″O¨). Permissions for sea lice collection were authorized by Marine Harvest S.A, Ruta 226, Km. 8, Camino El Tepual, Puerto Montt, Chile.

For the bioassays, deltamethrin (AlphaMax®) was prepared via serial dilutions with seawater to four concentrations (0, 1, 2, 3 ppb). A stock solution of 10 ppm was also prepared for each bioassay by diluting 1 ml of deltamethrin in 999 ml of seawater. In regards to azamethiphos (Bayer®), a stock solution of 1 ppm diluted in methanol and three serial dilutions with seawater to four concentrations (0, 1, 3, 10 ppb) were prepared. Ten sea lice adults (five females and five males) were exposed to each concentration of deltamethrin and azamethiphos using petri plates containing 50 ml of seawater (total individuals = 30). Each experiment was carried out in triplicate. The exposure period to either deltamethrin or azamethiphos was 40 and 30 min, respectively. During exposure, salmon lice were maintained at 12°C. After 24 h, the organisms were fixed in theRNAlater® RNA Stabilization Reagent (Ambion, USA) and stored at −80°C for subsequent RNA extraction. The protocols for bioassays were performed according to the SEACH Consortium (2006). All laboratory infections and culture procedures were carried out under guidelines approved by the ethics committee of the University of Concepción and under appropriate veterinary supervision.

High-throughput sequencing

The concentration 2 ppb and 3 ppb of deltamethrin or azamethiphos, respectively, was determined as EC50 in the bioassay. For this reason 15 females and 15 males of each group exposed to these concentrations, were used for MiSeq cDNA libraries preparation. Total RNA was extracted from pooled individuals for each sex (N = 10) using the RiboPure™ Kit (Ambion®, Life Technologies™, USA) following the manufacturer’s instructions. Quantity, purity, and quality of isolated RNA were measured in the TapeStation 2200 (Agilent Technologies Inc., Santa Clara, CA, USA) using the R6K Reagent Kit according to the manufacturer’s instructions; samples with RIN over 8.0 were used for library preparation. Subsequently, double-stranded cDNA libraries were constructed using the TruSeq RNA Sample Preparation Kit v2 (Illumina®, San Diego, CA, USA). Two biological replicates for each sample pool were sequenced by the MiSeq (Illumina®) platform using sequenced runs of 2x251 paired-end reads at the Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), University of Concepción, Chile. The cleaned short read sequences were deposited in the Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRX864101 and SRX864102 for deltamethrin and azamethiphos, respectively.

Sequence annotation and RNA-Seq analysis

From the EST-database generated for C. rogercresseyi [35], contigs were annotated using a database constructed from ABC transporter sequences described for D. pulex [12] and enriched with EST data for arthropods in order to determine putative gene descriptions. A cutoff E-value of 1E-05 was used.

The same EST database generated for C. rogercresseyi was used as a reference for RNA-Seq analysis. Using the CLC Genomic Workbench software, the reads obtained from female and male adult controls and individuals exposed to azamethiphos or deltamethrin were separately mapped against ABC transporter contigs. The RNA-Seq settings were a minimum length fraction = 0.6 and a minimum similarity fraction (long reads) = 0.5. The expression value was set as a reads per kilobase of exon model (RPKM). This normalized the number of reads to the size of assembled contigs and allowed for assessing the transcripts that were overexpressed among different groups. Furthermore, to compare differentiated transcript responses between sexes, the reads obtained for females and males were mapped separately over the ABC contigs using the RNA-Seq settings previously described [35]. A similar analysis was carried out between control and exposure groups using the new contigs obtained from de novo assembly as a reference. The metric distance was calculated using the Manhattan method, where the mean expression level in 5–6 rounds of k-means clustering was subtracted. Finally, a Kal’s statistical analysis test [36] was used to compare gene expression levels for larval stages and adults in terms of the log2 fold change (P = 0.0005; FDR corrected).

Amino acid sequence analyses

Protein alignments were conducted using MUSCLE, and phylogenetic trees were constructed using the neighbor-joining method with 1,000 bootstrap repetitions. Both analyses were carried out in Geneious 6.0.5 [37].

qPCR validation

Contig sequences of ABC subfamilies were obtained from the Illumina MiSeq database for C. rogercresseyi and used as a template for primer design with the Primer3 Tool [38] included in the Geneious Pro software [37] (Additional file 1: Table S1). For gene amplification, total RNA was isolated from sea louse exposed to 3 ppb of azamethiphos and 2 ppb of deltamethrin, using the TRI Reagent (Invitrogen, Carlsbad, CA, USA) protocol. The purity was determined (ratio A260/A280) with a Nanodrop ND1000 spectrophotometer (Thermo Fisher Scientific, Copenhagen, USA), and the integrity was determined by agarose gel under denaturant conditions. From 200 ng/μl of total RNA, cDNA was synthetized using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Glen Burnie, Maryland, USA). The qPCR runs were performed with StepOnePlus™ (Applied Biosystems, Life Technologies, USA) using the comparative ΔCt method. β-tubulin was selected as the housekeeping gene (HKG) [39]. Each reaction was conducted with a volume of 10 μL using the Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Scientific, USA). The amplification conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The data obtained were analyzed through the Kruskal-Wallis test with the Statistica software (Version 7.0, StatSoft, Inc.). Statistically significant differences were accepted with a p < 0.05.

SNPs mining and validation

Using the assembly obtained for all identified ABC transporters, SNPs mining was performed using the Genomics Workbench 5.0.1 software (CLC bio, Denmark). The parameters used were as follows: window length = 11, maximum gap and mismatch count = 2, minimum average quality of surrounding bases = 15, minimum quality of central base = 20, maximum coverage = 100, minimum coverage = 8, minimum variant frequency (%) = 35.0, and maximum expected variations (ploidy) = 2.

Results

Identification of ABC transporter subfamily genes from C. rogercresseyi

Through BLASTx analysis, 57 full or partial sequences were identified ABC transporter from transcriptome database described by Gallardo-Escárate et al. [35] and by using arthropod ABC transporter sequences described in public databases as a reference (Additional file 1: Table S2). Phylogenetic analysis revealed a relationship between seven C. rogercresseyi contigs and the ABCA subfamily described in D. pulex (Figure 1). From the seven contigs annotated for the ABCA subfamily, one of these presented an encoding sequence of 2,208 amino acids and the structure typical of this subfamily, with a large extracellular loop between the first two helices of the TMD (Table 1). BLASTp analysis demonstrated an identity of 39.8% and 39.3% with sequences described for Lottia gigantea (ESP01294) and Danio rerio (XP_005173137), respectively. Moreover, twelve contigs were annotated for the ABCB subfamily. Phylogenetic analysis grouped these sequences with the ABCA subfamily described for D. pulex (Figure 1). Contig 4,521 appeared as a full transporter encoding for 2,064 amino acids (Table 1). This also presented a 100% identity to the P-gp previously described in L. salmonis (ADT63773) and C. rogercresseyi (AHC54388). Thirteen contigs presented homology with the ABCC subfamily (Figure 1), with nine presenting high homology to ABCC1 transporters and four presenting homology with the ABCC4 subfamily (Additional file 1: Table S1). Also, two contigs presented high homology with sulfonylurea receptor (SUR) gene, member of ABCC subfamily (Additional file 1: Table S2). For the ABCD subfamily, four contigs were identified. Phylogenetic analysis grouped these sequences with orthologs described in D. pulex (Figure 1). BLASTp analysis demonstrated a 38.1% and 38.2% identity to ABCD4 described in Homo sapiens (NP_005041) and Mus musculus (NP_033018), respectively. From phylogenetic analysis, a relationship was observed between the ABCE and ABCF subfamilies of C. rogercresseyi (Figure 1). The sequences that were annotated to the ABCE subfamily presented an identity of 80.8% and 81.1% with homologs described in A. florea (XP_003690992) and Pediculus humanus (XP_002424051), respectively. The sequenced members of the ABCF subfamily presented an identity of 55.3% and 55.1% to sequences reported in A. aegypti (XP_001654470) and D. pulex (EFX69544), respectively. Furthermore, three contigs presented high homology with the ABCG subfamily (Additional file 1: Table S2) and were grouped with members of the ABCG subfamily described for D. pulex (Figure 1). Among these, contig 15272 encoded for haft transporters with the NBD-TMD domain traits (Table 1). Finally, three contigs of C. rogercresseyi were annotated as ABCH transporters (Figure 1). One of these encoded for 1,045 amino acids and presented an organization similar to that observed for ABCG transporters (Table 1).

Figure 1
figure 1

Phylogenetic relationships between ABC transporter subfamilies identified for C. rogercresseyi . The number at each node indicates the percentage of bootstrapping after 1,000 replications.

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Table 1 Amino acid position of conserved domain for ABC subfamily of C. rogercresseyi
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Transcriptomic profile of ABC transporters during larval stages and in adult C. rogercresseyi

RNA-Seq analysis was carried out in order to evaluate the transcriptome profiles of ABC transporters during the developmental stages of C. rogercresseyi (Figure 2). From the expression profiles in the larval nauplius I-II stages, the subfamilies ABCA/B/C/D were highly regulated. In the infective copepodid stage, the ABCC/E/F/G/H transporters were upregulated. The ABCD/E/F subfamilies were highly regulated in chalimus I-II stages, while in the chalimus III-IV stages, the ABCA/B/C/F subfamilies were upregulated (Figure 2). Furthermore, differences in expression profiles were observed between female and male C. rogercresseyi individuals. The ABCD/F subfamilies were upregulated in females, while in males, the ABCB/C subfamilies evidenced high regulation (Figure 2).

Figure 2
figure 2

Heat map generated for ABC transporter families identified from C. rogercresseyi transcriptome data reflecting gene expression values for all developmental stages.

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Transcriptomic profiles of ABC transporters in response to delousing drugs

RNA samples obtained from female and male C. rogercresseyi individuals exposed to 2 ppb of azamethiphos or 3 ppb of deltamethrin were sequenced using the MiSeq Illumina platform (Table 2). The sequencing runs of salmon lice exposed to azamethiphos yielded a total of 9.06 million reads for males and 10.4 million reads for females, with an average length of 223 and 254 bp, respectively. From adults exposed to deltamethrin, a total of 51.08 M and 27.54 M for males and females were generated, respectively, both with an average length of 219 bp.

Table 2 Statistical summary of Caligus rogercresseyi transcriptome following exposure to Azamethiphos or Deltamethrin
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Comparing the transcriptomic response of C. rogercresseyi adults exposed to azamethiphos, deltamethrin and the control group, the adults exposed to deltamethrin and the control group presented similar expression profiles of ABC transporters (Figure 3). The ABCB and ABCC subfamilies were upregulated in adult sea lice exposed to azamethiphos (Table 3). Also, increased expression was observed for both females and males in contigs annotated as members of the ABCA/D subfamilies in response to azamethiphos (Figure 4). An opposite effect was observed in contig 1285 annotated as an ABCF transporter, which was downregulated in sea lice exposed to azamethiphos. Furthermore, different transcriptomic responses were observed between sexes in response to azamethiphos, where the ABCB and ABCC subfamilies were only up-regulated in males. In response to deltamethrin, the ABCD/E/F subfamilies were highly regulated in adult sea lice (Table 3). Expression profiles of ABC transporters between females and males were similar (Figure 5). However, the contig 4421 annotated for ABCB transporter was upregulated in males exposed to deltamethrin, while females presented a high expression of contig 3679 annotated as a member of the ABCF subfamily.Additionally, in order to validate the transcription profiles obtained from sequencing data analysis, RT-qPCR was conducted on one represent of ABC transporters subfamily. The Pearson correlation evidenced a high linear dependence of fold change values obtained from both RNA-Seq and RT-qPCR for each ABC transporters in sea louse exposed to azamethiphos and deltamethrin (Figure 6).

Figure 3
figure 3

Cluster of gene expression levels between adult controls, adults exposed to 3 ppb of azamethiphos, and adults exposed to 2 ppb of deltamethrin.

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Table 3 Differential transcripts expression for ABC proteins subfamily en adults exposed to delousing drugs and control groups
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Figure 4
figure 4

Cluster of gene expression levels by sex between adult controls and adults exposed to 3 ppb of azamethiphos.

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Figure 5
figure 5

Cluster of gene expression levels by sex between adult controls and adults exposed to 2 ppb of deltamethrin.

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Figure 6
figure 6

Transcription expression validation for ABC transporters identified from C. rogercresseyi transcriptome in response to delousing drugs. Black circle: linear correlation among ABC transporters selected from RNA-seq and qPCR analysis after 3 ppb of azamethiphos exposure on sea lice. White circle: linear correlation among ABC transporters selected from RNA-seq and qPCR analysis after 2 ppb of azamethiphos exposure on sea lice.

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Single nucleotide polymorphism (SNP) mining

A total of 52 SNPs were identified in 17 contigs that annotated for ABC transporter sequences in C. rogercresseyi (Additional file 1: Table S3). For contigs annotated for the ABCA subfamily, a non-synonymous variation was identified in the TMD2 domain (Additional file 1: Table S3). Three contigs presented SNPs variations for the ABCB subfamily, one of which, contig 9296, had a non-synonymous variation in the NBD1 domain (Additional file 1: Table S3). For the ABCC subfamily, seven contigs presented SNPs in the open reading frame and 3′UTR regions (Additional file 1: Table S3). In ABCD/E/G/H subfamilies, the identified variations were synonymous or were present in the 3′UTR region (Additional file 1: Table S3). Nevertheless, more studies are necessary to determinate the function of non-synonymous variations in ABC transporters and the possible association of these with delousing drug resistance.

Discussion

The ABC family is composed of proteins that include a wide range of compounds both within and exterior to the cell. In eukaryotes, many of the functions that these proteins have in the cell remain unknown. However, in Drosophila melanogaster these proteins have been observed to participate in the modulation of molting hormones such as the ecdysteroids. These transporters have also been found in insects to play a role in insecticide tolerance [16,40]. Currently in arthropods, 64, 56, and 46 ABC proteins have been identified in D. pulex [12], Drosophila [16], and T. japonicus [23], respectively. The present study found 57 contigs with homology to the different ABC proteins subfamilies during distinct larval and adult stages of C. rogercresseyi development [35]. Of these contigs, members belonging to eight of the ABC subfamilies (A-H) described in arthropods were identified [16].

Differences in the expression of the eight ABC proteins subfamilies were evaluated in silico during the distinct developmental stages of C. rogercresseyi. The ABCA subfamily was upregulated during the nauplius I-II and chalimus III-IV larval stages. The physiological function of this transporter subfamily is still unclear in arthropod organisms [41]. However, in T. castaneium, blocking this subfamily causes mortality in pupae and adults [21]. Taking the results obtained in T. castaneum together with the greater expression levels observed in juvenile C. rogercresseyi, it is possible to suggest that the ABCA subfamily plays a role during early ontogenetic development.

In turn, the ABCB subfamily has been widely studied in humans due to its function as a MRP in studies related to the control of cancer [42,43]. Moreover, this subfamily has been implicated in the molting process and the developmental transition from pupa to adult in T. castaneum [21]. The present study found greater expression levels in juvenile chalimus III-IV stages and in adult C. rogercresseyi, which is a result similar to that observed in T. castaneum [21] and T. japonicus [23]. Taken together, these findings indicate greater metabolic activity in response to pharmaceutical treatments during the parasitic stages of C. rogercresseyi.

Similar to the ABCB subfamily, ABCC transporters have detoxifying activities and present specific binding sites to drugs (MRPs) [13]. In C. rogercresseyi, transporters associated with ABCC1 and ABCC4 were identified and overexpressed throughout the course of the nauplius I-II larval stages. The ABCC1 transporters are considered “long” MRPs and present an extra transmembrane domain in the N-terminal termed TMD0. This is compared to “short” MRPs, among which is ABCC4 [44]. Long MRPs, or MRP1, have been associated with xenobiotic resistance in humans [45], T.ni [28], and L. salmonis [31]. Other ABC protein member of this subfamily is the sulfonylurea receptor (SUR). In arthropod SUR proteins have been related to chitin synthesis and in some reports suggest that SUR are putative targets for some insecticide [16,46]. For C. rogercresseyi some contigs with high homology with SUR gene were highly regulated in larval stages, which can be related to the sea lice cuticle biogenesis. On the other hand, the high transcript levels observed during the larval stages of C. rogercresseyi allow for associating the C subfamily, in addition to its detoxifying characteristics, to processes related to development and maturation during early developmental stages, as has been observed in other invertebrates [16,47].

The ABCD subfamily in C. rogercresseyi was overexpressed in the nauplius I-II and chalimus I-II larval stages. In contrast to other transporter types, this subfamily is found in membrane-bound peroxisome, and its function is to transport fatty acids to the interior of this organelle [16,41,48]. These transporters have also been observed to play a role in the metabolism and development of arthropods [49]. Congruent with that observed for C. rogercresseyi, this transporter subfamily has been found expressed during all of the developmental stages in T. japonicus [23].

From the infective copepodid stages, an increased expression was observed for the ABCE and ABCF subfamilies in C. rogercresseyi. In humans, the ABCE1 transporter has inhibitory actions on RNase L, an important enzyme related to interferon functions in response to the presence of a virus [48]. It has also been found that blocking the ABCE-F transporters in T. castaneum causes mortality in the pre-pupa stage, thereby impeding development into adults [21]. Moreover, the ABCE transporter presents ubiquitous expression during all developmental stages in T. japonicus [23].

The ABCG subfamily of transporters corresponds to the haft transporter and presents inverted domains in the extreme C-terminal [12]. In C. rogercresseyi, this subfamily was principally overexpressed during the nauplius II larval stage. The ABCG transporter is homologous to the white protein that forms heterodimers in D. melanogaster with the brown and scarlet proteins, which themselves act as a precursor to pigmentation [50]. In T. castaneum, the inactivation of this gene provoked an arrest of development during the pre-pupa stages [21]. The ABCG transporters have also been found to induce the expression of genes linked to the molting hormone 20-ecdysone (20E) in D. melanogaster [51]. Given the expression of this transporter during the nauplius II stage in C. rogercresseyi, it could be associated with development towards the copepodid stage.

The final subfamily identified was the ABCH subfamily, which presents a domain formation similar to that of the ABCG subfamily [12]. In C. rogercresseyi, three contigs were identified that annotated for the ABCH subfamily, and these were overregulated in the nauplius II stage. Despite that the function of the ABCH transporters is still unclear, in T. cantaneum these have been found to play a fundamental role in the transport of lipids to the cuticle, thereby generating a hydrophobic barrier for the organism [21]. On the other hand, in T. urticae these types of transporters could be involved in the process of diapause [52].

The ABC transporters have been widely studied for their role as MRPs, especially the ABCB and ABCC subfamilies. The ABC transporters have been found to participate in the detoxification of pyrethroids and avermectins, among other chemicals generally used in invertebrate pest control [53-55]. For example, increased transcript levels of the ABCB1 (P-gp) transporter in L. salmonis has been linked to generated resistance to EMB [10,11]. Similarly, an increased gene expression of ABC transporters in A. aegypti populations resistant to pyrethroids suggests the participation of these transporters in detoxifying processes [29]. In addition to pyrethroids, the effect of organophosphates on expression levels of ABC transporters has been evaluated in rats, showing that exposure to diazinon induced an increased intestinal expression of P-gp [56]. Moreover, in resistant strains of Rhipicephalus microplus treated with an inhibitor of ABCB transporters and exposed to different concentrations of chlorpyrifos, the lethal concentration of this organophosphate was reduced as compared to strains not treated with an inhibitor. These findings reveal the interaction between ABC transports and the detoxification process of organophosphates [53].

The present study evaluated the transcriptomic response of the ABC transporters identified in adult C. rogercresseyi individuals exposed to the pyrethroid deltamethrin and the organophosphate azamethiphos. For both treatments, the ABCB and ABCC transporter subfamilies presented higher expression levels in salmon lice exposed to the pharmaceuticals. Additionally, differences in expression profiles were observed between males and females, a result which has also been obtained in EBM resistant strains of L. salmonis [57]. In regards to the ABCG subfamily, these have been found to transport drugs and sterols [16], and in P. xylostella resistant to insecticides, an increased expression of the ABCH transporters has been reported, thus suggesting a possible detoxifying role [58]. However, in C. rogercresseyi, no significant change in transcript levels of these two subfamilies was found in adults exposed to deltamethrin or azamethiphos.

The presence of mutations in genes coding for the ABC transporters can generate changes in molecular functions and related biological processes. For example, mutations in human ABCD transporters have been found to cause adrenal insufficiency and the demyelination of neurons [59]. Moreover, variations in transporters linked to processes of detoxification can induce resistance in organisms, such as with the insertion of tyrosine in the ABCC4 transporter of Bombyx mori, where strains with this insertion were resistant to the toxin Cry1Ab [60]. Another possible mutation is through SNP variations, which could be an important tool in identifying organisms resistant or susceptible to certain drugs. For example, 13 polymorphisms have been identified in humans in distinct ABC transporter subfamilies, and these have led to associations between individual responses and distinct therapeutic drug treatments [61]. Apart from this, the nematode Onchocerca volvulus presents alterations in P-gp transporter functions in association with the presence of SNPs that generate non-synonymous changes [62]. The present study identified 17 ABC transporters related to polymorphisms in a single nucleotide. The identified SNPs evidenced both synonymous and non-synonymous mutations in conserved domains and UTR regions. Future studies will focus on relating the reported mutations with the response of C. rogercresseyi to the distinct pharmaceuticals used in infestation control.

Conclusions

The present study applied genomic approaches to identify 57 sequences annotating for the eight members of the ABC transporter subfamilies in C. rogercresseyi. The ABC transporters were evaluated in silico and demonstrated changes in expression throughout the developmental stages of the salmon louse. Additionally, the expression profiles of ABC transporters in adult individuals exposed to deltamethrin or azamethiphos were evaluated, finding increased expression levels for the ABCB and ABCC subfamilies. Finally, mutations were found in 17 ABC transporters, with these being located in both the open reading frame and UTR regions. Future studies will evaluate the effects that the identified SNPs have in C. rogercresseyi strains resistant or susceptible to the drugs used for its control in the Chilean salmon aquaculture industry.

References

  1. Bravo S. Sea lice in Chilean salmon farms. Aquaculture. 2003;23:197–200.

    Google Scholar 

  2. Costello MJ. Ecology of sea lice parasitic on farmed and wild fish. Trends Parasitol. 2006;22:475–83.

    Article  PubMed  Google Scholar 

  3. Hamilton-West C, Arriagada G, Yatabe T, Valdés P, Hervé-Claude LP, Urcelay S. Epidemiological description of the sea lice (Caligus rogercresseyi) situation in southern Chile in August 2007. Prev Vet Med. 2011;104:341–5.

    Article  PubMed  Google Scholar 

  4. Oelckers K, Vike S, Duesund H, Gonzalez J, Wadsworth S, Nylund A. Caligus rogercresseyi as a potential vector for transmission of Infectious Salmon Anaemia (ISA) virus in Chile. Aquaculture. 2014;420‚Äì421(0):126–32.

    Article  Google Scholar 

  5. Torrissen O, Jones S, Asche F, Guttormsen A, Skilbrei OT, Nilsen F, et al. Salmon lice – impact on wild salmonids and salmon aquaculture. J Fish Dis. 2013;36(3):171–94.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Cárcamo JG, Aguilar MN, Barrientos C, Carreño CF, Quezada C, Bustos C, et al. Effect of emamectin benzoate on transcriptional expression of cytochromes P450 and the multidrug transporters (Pgp and MRP1) in rainbow trout (Oncorhynchus mykiss) and the sea lice Caligus rogercresseyi. Aquaculture. 2011;321:207–15.

    Article  Google Scholar 

  7. Bravo S, Treasurer J, Sepulveda M, Lagos C. Effectiveness of hydrogen peroxide in the control of Caligus rogercresseyi in Chile and implications for sea louse management. Aquaculture. 2010;303:22–7.

    Article  CAS  Google Scholar 

  8. Grant AN. Medicines for sea lice. Pest Manag Sci. 2002;58(6):521–7.

    Article  CAS  PubMed  Google Scholar 

  9. Jones M, Sommerville C, Wootten R. Reduced sensitivity of the salmon louse, Lepeophtheirus salmonis, to the organophosphate dichlorvos. J Fish Dis. 1992;15(2):197–202.

    Article  Google Scholar 

  10. Heumann J, Carmichael S, Bron JE, Tildesley A, Sturm A. Molecular cloning and characterisation of a novel P-glycoprotein in the salmon louse Lepeophtheirus salmonis. Comp Biochem Physiol, Part C: Toxicol Pharmacol. 2012;155(2):198–205.

    CAS  Google Scholar 

  11. Igboeli O, Purcell S, Wotton H, Poley J, Burka J, Fast M. Immunostimulation of Salmo salar L., and its effect on Lepeophtheirus salmonis (Kroyer) P-glycoprotein mRNA expression following subsequent emamectin benzoate exposure. J Fish Dis. 2013;36(3):339–51.

    Article  CAS  PubMed  Google Scholar 

  12. Sturm A, Cunningham P, Dean M. The ABC transporter gene family of Daphnia pulex. BMC Genomics. 2009;10(1):170.

    Article  PubMed Central  PubMed  Google Scholar 

  13. Dean M, Hamon Y, Chimini G. The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res. 2001;42(7):1007–17.

    CAS  PubMed  Google Scholar 

  14. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8(1):67–113.

    Article  CAS  PubMed  Google Scholar 

  15. Hollenstein K, Dawson RJP, Locher KP. Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol. 2007;17(4):412–8.

    Article  CAS  PubMed  Google Scholar 

  16. Dermauw W, Van Leeuwen T. The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance. Insect Biochem Mol Biol. 2014;45:89–110.

    Article  CAS  PubMed  Google Scholar 

  17. Davidson AL, Chen J. ATP-binding cassette transporters in bacteria. Annu Rev Biochem. 2004;73(1):241–68.

    Article  CAS  PubMed  Google Scholar 

  18. Roth CW, Holm I, Graille M, Dehoux P, Rzhetsky A, Wincker P, et al. Identification of the Anopheles gambiae ATP-binding cassette transporter superfamily genes. Mol Cells. 2003;15(2):150–8.

    CAS  PubMed  Google Scholar 

  19. Liu S, Zhou S, Tian L, Guo E, Luan Y, Zhang J, et al. Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori. BMC Genomics. 2011;12(1):491.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Xie X, Cheng T, Wang G, Duan J, Niu W, Xia Q. Genome-wide analysis of the ATP-binding cassette (ABC) transporter gene family in the silkworm, Bombyx mori. Mol Biol Rep. 2012;39(7):7281–91.

    Article  CAS  PubMed  Google Scholar 

  21. Broehan G, Kroeger T, Lorenzen M, Merzendorfer H. Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum. BMC Genomics. 2013;14(1):6.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Dermauw W, Osborne EJ, Clark RM, Grbić M, Tirry L, Van Leeuwen T. A burst of ABC genes in the genome of the polyphagous spider mite Tetranychus urticae. BMC Genomics. 2013;14(1):317.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Jeong C-B, Kim B-M, Lee J-S, Rhee J-S. Genome-wide identification of whole ATP-binding cassette (ABC) transporters in the intertidal copepod Tigriopus japonicus. BMC Genomics. 2014;15(1):651.

    Article  PubMed Central  PubMed  Google Scholar 

  24. Szakács G, Annereau J-P, Lababidi S, Shankavaram U, Arciello A, Bussey KJ, et al. Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells. Cancer Cell. 2004;6(2):129–37.

    Article  PubMed  Google Scholar 

  25. Massey P, Fojo T, Bates S. ABC Transporters: Involvement in Multidrug Resistance and Drug Disposition. In: Rudek MA, Chau CH, Figg WD, McLeod HL, editors. Handbook of Anticancer Pharmacokinetics and Pharmacodynamics. New York: Springer; 2014. p. 373–400.

    Chapter  Google Scholar 

  26. Juranka PF, Zastawny RL, Ling V. P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins. FASEB J. 1989;3(14):2583–92.

    CAS  PubMed  Google Scholar 

  27. Luckenbach T, Epel D. ABCB-and ABCC-type transporters confer multixenobiotic resistance and form an environment-tissue barrier in bivalve gills. Am J Physiol Regul Integr Comp Physiol. 2008;294(6):R1919–29.

    Article  CAS  PubMed  Google Scholar 

  28. Simmons J, D’Souza O, Rheault M, Donly C. Multidrug resistance protein gene expression in Trichoplusia ni caterpillars. Insect Mol Biol. 2013;22(1):62–71.

    Article  CAS  PubMed  Google Scholar 

  29. Bariami V, Jones CM, Poupardin R, Vontas J, Ranson H. Gene Amplification, ABC Transporters and Cytochrome P450s: Unraveling the Molecular Basis of Pyrethroid Resistance in the Dengue Vector, Aedes aegypti. PLoS Negl Trop Dis. 2012;6(6):e1692.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Valenzuela-Muñoz V, Nuñez-Acuña G, Gallardo-Escárate C. Molecular characterization and transcription analysis of P-glycoprotein gene from the salmon louse Caligus rogercresseyi. J Aquaculture Res Dev. 2014;5(236): doi:10.4172/2155-9546.1000236

  31. Heumann J, Carmichael SN, Bron JE, Sturm A. Isolation and characterisation of four partial cDNA sequences encoding multidrug resistance-associated proteins (MRPs) in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837). Aquaculture. 2014;424–425:207–14.

    Article  Google Scholar 

  32. Bravo S, Sevatdal S, Horsberg TE. Sensitivity assessment of Caligus rogercresseyi to emamectin benzoate in Chile. Aquaculture. 2008;282(1‚Äì4):7–12.

    Article  CAS  Google Scholar 

  33. Bravo S, Sepulveda M, Silva MT, Costello MJ. Efficacy of deltamethrin in the control of Caligus rogercresseyi (Boxshall and Bravo) using bath treatment. Aquaculture. 2014;432:175–80.

    Article  CAS  Google Scholar 

  34. Helgesen KO, Bravo S, Sevatdal S, Mendoza J, Horsberg TE. Deltamethrin resistance in the sea louse Caligus rogercresseyi (Boxhall and Bravo) in Chile: bioassay results and usage data for antiparasitic agents with references to Norwegian conditions. J Fish Dis. 2014;37(10):877–90.

    Article  CAS  PubMed  Google Scholar 

  35. Gallardo-Escárate C, Valenzuela-Muñoz V, Núñez-Acuña G. RNA-Seq analysis using de novo transcriptome assembly as a reference for the salmon louse Caligus rogercresseyi. Plos One. 2014;9(4):e92239.

    Article  PubMed Central  PubMed  Google Scholar 

  36. Kal AJ, van Zonneveld AJ, Benes V, van den Berg M, Koerkamp MG, Albermann K, et al. Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources. Mol Biol Cell. 1999;10(6):1859–72.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Drummond A. Geneious. v. 5.1 Available from: http://www.geneious.com. 2009.

  38. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.

    CAS  PubMed  Google Scholar 

  39. Gallardo-Escárate C, Valenzuela-Muñoz V, Nuñez-Acuña G, Chávez-Mardones J, Maldonado-Aguayo W. Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi. Gene. 2014;536(1):1–8.

    Article  PubMed  Google Scholar 

  40. Jones CM, Toé HK, Sanou A, Namountougou M, Hughes A, Diabaté A, et al. Additional selection for insecticide resistance in urban malaria vectors: DDT resistance in Anopheles arabiensis from Bobo-Dioulasso, Burkina Faso. PLoS One. 2012;7(9):e45995.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Strauss A, Wang D, Stock M, Gretscher R, Groth M. Tissue-Specific Transcript Profiling for ABC Transporters in the Sequestering Larvae of. 2014.

    Google Scholar 

  42. Gillet J-P, Gottesman MM. Mechanisms of multidrug resistance in cancer. Springer. 2010;596:47–61.

    CAS  Google Scholar 

  43. Natarajan K, Xie Y, Baer MR, Ross DD. Role of breast cancer resistance protein (BCRP/ABCG2) in cancer drug resistance. Biochem Pharmacol. 2012;83(8):1084–103.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Deeley R, Westlake C, Cole S. Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev. 2006;86:849–99.

    Article  CAS  PubMed  Google Scholar 

  45. Cole S, Bhardwaj G, Gerlach J, Mackie J, Grant C, Almquist K, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science. 1992;258(5088):1650–4.

    Article  CAS  PubMed  Google Scholar 

  46. Abo-Elghar GE, Fujiyoshi P, Matsumura F. Significance of the sulfonylurea receptor (SUR) as the target of diflubenzuron in chitin synthesis inhibition in Drosophila melanogaster and Blattella germanica. Insect Biochem Mol Biol. 2004;34(8):743–52.

    Article  CAS  PubMed  Google Scholar 

  47. Roepke TA, Hamdoun AM, Cherr GN. Increase in multidrug transport activity is associated with oocyte maturation in sea stars. Dev Growth Differ. 2006;48(9):559–73.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Kashiwayama Y, Seki M, Yasui A, Murasaki Y, Morita M, Yamashita Y, et al. 70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins. Exp Cell Res. 2009;315(2):190–205.

    Article  CAS  PubMed  Google Scholar 

  49. Theodoulou FL, Holdsworth M, Baker A. Peroxisomal ABC transporters. FEBS Lett. 2006;580(4):1139–55.

    Article  CAS  PubMed  Google Scholar 

  50. Dreesen T, Johnson D, Henikoff S. The brown protein of Drosophila melanogaster is similar to the white protein and to components of active transport complexes. Mol Cell Biol. 1988;8(12):5206–15.

    PubMed Central  CAS  PubMed  Google Scholar 

  51. Liu H, Jia Q, Tettamanti G, Li S. Balancing crosstalk between 20-hydroxyecdysone-induced autophagy and caspase activity in the fat body during Drosophila larval-prepupal transition. Insect Biochem Mol Biol. 2013;43(11):1068–78.

    Article  CAS  PubMed  Google Scholar 

  52. Bryon A, Wybouw N, Dermauw W, Tirry L, Van Leeuwen T. Genome wide gene-expression analysis of facultative reproductive diapause in the two-spotted spider mite Tetranychus urticae. BMC Genomics. 2013;14(1):815.

    Article  PubMed Central  PubMed  Google Scholar 

  53. Pohl P, Klafke G, Júnior JR, Martins JR, Silva Vaz Jr I, Masuda A. ABC transporters as a multidrug detoxification mechanism in Rhipicephalus (Boophilus) microplus. Parasitol Res. 2012;111(6):2345–51.

    Article  PubMed  Google Scholar 

  54. James CE, Davey MW. Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans. Int J Parasitol. 2009;39(2):213–20.

    Article  CAS  PubMed  Google Scholar 

  55. J-FB P, FB L’H, Liu Z, Prichard RK, Georges E. Reversal of P-glycoprotein-associated multidrug resistance by ivermectin. Biochem Pharmacol. 1997;53(1):17–25.

    Article  Google Scholar 

  56. Lecoeur S, Videmann B, Mazallon M. Effect of organophosphate pesticide diazinon on expression and activity of intestinal P-glycoprotein. Toxicol Lett. 2006;161(3):200–9.

    Article  CAS  PubMed  Google Scholar 

  57. Igboeli OO, Burka JF, Fast MD. Sea lice population and sex differences in P-glycoprotein expression and emamectin benzoate resistance on salmon farms in the Bay of Fundy, New Brunswick, Canada. Pest Manag Sci. 2014;70:905–14.

    Article  CAS  PubMed  Google Scholar 

  58. You M, Yue Z, He W, Yang X, Yang G, Xie M, et al. A heterozygous moth genome provides insights into herbivory and detoxification. Nat Genet. 2013;45(2):220–5.

    Article  CAS  PubMed  Google Scholar 

  59. Petriv I, Pilgrim DB, Rachubinski RA, Titorenko VI. RNA interference of peroxisome-related genes in C. elegans: a new model for human peroxisomal disorders. Physiol Genomics. 2002;10(2):79–91.

    Article  CAS  PubMed  Google Scholar 

  60. Atsumi S, Miyamoto K, Yamamoto K, Narukawa J, Kawai S, Sezutsu H, et al. Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori. Proc Natl Acad Sci. 2012;109(25):E1591–8.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  61. Iida A, Saito S, Sekine A, Mishima C, Kitamura Y, Kondo K, et al. Catalog of 605 single-nucleotide polymorphisms (snps) among 13 genes encoding human atp-binding cassette transporters: abca4, abca7, abca8, abcd1, abcd3, abcd4, abce1, abcf1, abcg1, abcg2, abcg4, abcg5, and abcg8. J Hum Genet. 2002;47(6):285–310.

    Article  CAS  PubMed  Google Scholar 

  62. Bourguinat C, Ardelli BF, Pion SDS, Kamgno J, Gardon J, Duke BOL, et al. P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus. Mol Biochem Parasitol. 2008;158(2):101–11.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was funded through CONICYT-Chile, FONDAP project grant 15110027. The authors also give thanks to Marine Harvest Chile S.A.

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Authors and Affiliations

  1. Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), University of Concepción, PO. Box 160-C, Concepción, Chile

    Valentina Valenzuela-Muñoz & Cristian Gallardo-Escárate

  2. Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, Scotland, UK

    Armin Sturm

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  1. Valentina Valenzuela-Muñoz
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Correspondence to Cristian Gallardo-Escárate.

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The authors declare that they have no competing interests.

Authors’ contributions

CG-E conceived the idea, performed the experimental analyses collected the samples; VV-M performed the sequencing runs; VV-M and CG-E performed the bioinformatic analyses; VV-M, AS, CG-E led the manuscript’s draft preparation. All authors have read and approved the final version of this manuscript.

Additional file

Additional file 1: Table S1.

Primer list for ABC transporters identified in C. rogercresseyi for qPCR validation. Table S2. BLASTx analysis of 57 C. rogercresseyi ABC proteins. Table S3. List of SNPs identified for ABC proteins from C. rogercresseyi.

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Valenzuela-Muñoz, V., Sturm, A. & Gallardo-Escárate, C. Transcriptomic insights on the ABC transporter gene family in the salmon louse Caligus rogercresseyi . Parasites Vectors 8, 209 (2015). https://doi.org/10.1186/s13071-015-0801-x

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Parasites & Vectors

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