Molecular Characterization and Transcription Analysis of P-Glycoprotein Gene from the Salmon Louse Caligus rogercresseyi

One of the most important defense mechanisms of ectoparasites against chemotherapeutic or xenobiotic agents is related with the ATPbinding cassette (ABC) transporter family [1] he ABC transporters have been associated to multidrugs resistance (MDR) in different organisms. ABC proteins are able to recognize different chemical substrates and to control their transport on a cellular level [2]. Through this, the ABC proteins have a pivotal role in blocking high concentrations neurotoxins, which intend to enter into the nervous system of parasitic invertebrates [3,4]. Within this family of proteins are the multidrug resistance-associated protein, the half-transporters, and the P-glycoprotein (Pgp). Pgp is a highly conserved membranebound protein in eukaryotes and prokaryotes, and it functions as an ATP-dependent efflux pump, reducing the concentration of drugs in the cells and conferring resistance [5-7]. In mammals, Pgp has been reported as one of the principal barriers against the entrance of drugs from the blood stream into the nervous system [8].


Introduction
One of the most important defense mechanisms of ectoparasites against chemotherapeutic or xenobiotic agents is related with the ATPbinding cassette (ABC) transporter family [1] he ABC transporters have been associated to multidrugs resistance (MDR) in different organisms. ABC proteins are able to recognize different chemical substrates and to control their transport on a cellular level [2]. Through this, the ABC proteins have a pivotal role in blocking high concentrations neurotoxins, which intend to enter into the nervous system of parasitic invertebrates [3,4]. Within this family of proteins are the multidrug resistance-associated protein, the half-transporters, and the P-glycoprotein (Pgp). Pgp is a highly conserved membranebound protein in eukaryotes and prokaryotes, and it functions as an ATP-dependent efflux pump, reducing the concentration of drugs in the cells and conferring resistance [5][6][7]. In mammals, Pgp has been reported as one of the principal barriers against the entrance of drugs from the blood stream into the nervous system [8].
Caligus rogercresseyi [9] constitutes the main cause of economical losses in farmed salmon Chilean industry. Infection by the salmon louse does not generally cause host death, but its interaction with the salmon causes a high level of stress which depresses the immune system and increases susceptibility to bacterial and viral pathogens [10,11]. In order to control the prevalence of C. rogercresseyi, xenobiotics like pyrethroids [11], organophosphates [12], and avermectins [13] have been used. The overuse of these chemical agents has been shown to diminish their efficiency in treating parasite infestations due to resistance [14][15][16]. The time required by the parasite to acquire resistance depends on various factors, among which are environmental conditions and the concentration and frequency of the treatment [12,[17][18][19]. Numerous studies have shown behavioral differences of the salmon louse when it is exposed to parasiticides depending on its developmental [20][21][22]. Avermectins used for the salmon louse control, such as emamectin benzoate (EMB), have been shown to be effective during the development stages of copepods [23]. Herein, Pgp has been observed to be the main defense of the salmon louse against xenobiotics, particularly to EMB, which joins to the gamma-aminobutryric acid (GABA) receptors, causing paralysis [24]. The authors Heumann et al. [17] identified Pgp in Lepeophtheirus salmonis [18] that were overexpressed in resistant strain after 24 h of exposure to EMB, suggesting their participation in the response to the antiparasite. Other xenobiotic used for sea lice control is the pyrethroid named deltamethrin (Alphamax ® ). Deltamethrin is applied by topical treatments and interact with sodium channel depolarized the nerve ending [26]. Similarly, it has been observed that the family of cytochrome P450 enzymes participates in pyrethroid detoxification process in insects [27][28][29].
In Chile, the most widely used xenobiotic for the salmon lice control is deltamethrin [30]. However, there are no studies addressed to evaluate the transcription expression of Pgp from salmon louse exposed to deltamethrin. Moreover, studies are limited in regards to the functions that detoxification genes like Pgp have during the development of salmon lice, and it is only possible to find comparative studies on the effects that EMB has on expression levels in adult copepods. Given this, the aims of the present study were to perform a molecular characterization of the P-glycoprotein in Caligus rogercresseyi, evaluate their expression in adults treated with deltamethrin and assay its expression on a transcriptomic level during larval and adult stages.

Samples
Samples from the copepodid and chalimus stages and of adult male and female sea lice were collected from a commercial farm located in region X in Chile (41°40'48.5"S; 73°02'31.34"O¨). The permissions for the sea lice collections were authorized by Marine Harvest S.A, Ruta 226, Km. 8, Camino El Tepual, Puerto Montt, Chile. The specimens were used to characterize the Pgp mRNA. A pool of each stage was fixed in RNAlater ® RNA Stabilization Reagent (Ambion, USA) and stored at -80ºC for subsequent RNA extraction.

Bioassay
Formulations of deltamethrin were made using "AlphaMax ® " (10 mg deltamethrin/mL) diluted with seawater to six concentrations (0, 1, 2, 3 ppb). A stock solution of 10 ppm was prepared for each bioassay by diluting 1 ml AlphaMax in 999 ml seawater. The other concentrations were made by serial dilutions. Thirty adults (fifteen females and five males) of sea louse were exposed to each concentration of the deltamethrin formulation in petri plate containing 50 ml of seawater (three biological replicates were also evaluated). The exposure period was 40 min according to the recommended treatment time for AlphaMax. During the exposure, organisms were maintained at 12°C. After 24 h the sea louse were fixed in RNAlater ® RNA Stabilization Reagent (Ambion, USA) and stored at -80ºC for subsequent RNA extraction.

P-glycoprotein gene isolation
A partial sequence of the Pgp gene was identified from a C. rogercresseyi EST-database from Illumina MiSeq library generated for C. rogercresseyi in the Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), University of Concepción. The sequence was used as a template for primer design with the Primer3 tool [30] (Table 1) included in the Geneious Pro software [31]. For gene amplification, total RNA was isolated 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). PCR analysis was performed using 1 μl of cDNA, 10 µM of each primer (Table 1), 1.5 mM MgCl 2 , and 0.06 U taq DNA polymerase (Thermo Scientific, Maryland, USA). PCR was performed in 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s. The PCR product was observed by electrophoresis on 1% agarose gel and sequenced in the ABI 3730xl sequencer (Applied Biosystems, CA, USA). The sequence was analyzed using the Geneious 5.1.6 software [31]. The resulting partial sequence, termed Cr-Pgp, was used for designing specific new primers (Table 1)

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 5.1.6 [31]. For identifying the conserved domains and active sites, the PROSITE [32] and SMART [33] platforms were used.

SNPs mining and validation
Using the assembly obtained for the Pgp sequence, 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.
For SNPs validation, high resolution melting analysis (HRMA) was performed using primers designed with the Primer3 tool [30] ( Table 1) included in the Geneious Pro software [31]. Total DNA was isolated using the E.Z.N.A DNA extraction kit (Omega) from 20 adult salmon lice (10 females and 10 males). The PCR was carried out in 10 µl reaction with 13 ng of template DNA using the Fast EvaGreen ® qPCR Master Mix (Biotum). For HRMA, thermal cycling was performed with an ECO Real Time PCR System (Ilumina Inc, USA) as follows: 2 min for enzyme activation, 40 cycles at 95°C for 5 s, 56°C for 5 s, and 60°C for 25 s. HRMA data were collected between 60 and 95°C with a temperature interval of 0.3%. Genotyping was analyzed for the presence of a discrete melting curve using the software EcoReal Time System (Illumina Inc. USA).

Expression profiles of Cr-Pgp
Transcripts levels were evaluated for the copepodid and chalimus stages and for adult females and males of C. rogercresseyi. On other hand, an expression level of Cr-Pgp in sea lice exposed to deltamethrin were evaluated and compared with P450 gene expression. The primers

Molecular characterization of Cr-P-glycoprotein
The sequence for the characterized Pgp gene was obtained by assembling partial sequences identified in the Illumina cDNA library for C. rogercresseyi and the fragment amplified by RACE-PCR. The full  Figure 1A). The amino acid sequence had a predicted molecular weight of 140.3 kDa and an isoelectric point of 6.07. ABC transporters minimally consist of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). These can be found on the same protein or on two different ones. Herein, the translation of the Cr-Pgp cDNA sequence detected ABC-transporter transmembrane domains (TMDs) and nucleotide binding domains (NBD), arranged in an N-terminal to C-terminal order of TMD-NBD-TMD-NBD, the typical domain architecture of ABC full transporters ( Figure 1A). Further, two TMDs with six transmembrane helices each, and two cytosolic domains were predicted.
From a multiple sequence alignment of Cr-Pgp, a 75.7% identity was observed with sequences described for Lepeophtheirus salmonis and a 43.3% identity was observed with the homologues sequences described for Homo sapiens and Drosophila menalogaster ( Figure 1B). After performing a phylogenetic analysis through the neighbor-joining method, two clades were observed, one of these being for vertebrates and the other for invertebrate species. The Cr-Pgp sequence was grouped, as expected, within the sequences of Cr-Pgp reported for L. salmonis (Figure 2).

Expression analysis
To determine the effect of deltamethrin over Cr-Pgp expression we evaluated individuals obtained from bioassays and compared with a gene involved in detoxification process as P450. From the qPCR analysis higher expression levels of Cr-Pgp and Cr-P450 were observed in samples exposed to 2 ppb of deltamethrin ( Figure 3). Furthermore, Cr-Pgp in female shows higher transcription levels than males, which is reflected in the higher percentage of survival in female individuals (Table 2). Contrary, Cr-P450 not significant differences in gene expression between male and female samples were observed (Figure 3). Through qPCR, the transcript levels for the characterized gene were analyzed in different stages of development copepodid, chalimus, and male and female adults. Relative expression calculated using housekeeping gene beta-tubulin presented a stability value as calculated by the NormFinder algorithm. Comparing the four samples, the highest expression levels of Cr-Pgp were observed in adult females, and an overregulation was registered for adult males ( Figure  4). Comparing the copepodid and chalimus stages, an up-regulation     was observed in chalimus samples. The expression analysis showed that Cr-Pgp it was strongly regulated in females, suggesting a better detoxification mechanism to pyrethroid.

SNPs identification and validation
From the characterization of nucleotide sequence, a total of eleven SNPs present in the ORF of Cr-Pgp obtained from 10,921 reads were identified. Two SNPs located at +1,266A/T and +1,353 G/A were nonsynonymous and involved in amino acid changes between Met/ Leu and Val/Ile (Table 3). Furthermore, 20 adults of C. rogercresseyi were genotyped by HRMA analysis and showed three profiles of melt curves, evidencing the different allelic variants for these SNPs ( Figure  5). However, it is necessary evaluates the SNPs in groups with different susceptibility to pyrethroids in order to associate putative mutations with resistance to xenobiotics.

Discussion
ABC transporters are transmembrane proteins responsible for the translocation of various compounds in prokaryotic and eukaryotic organisms [8]. In mammals and fish, proteins belonging to this family have a role in the metabolism of xenobiotics [8]. In bivalves which are constantly exposed to toxins, the proteins of this family have been observed to act as a protection barrier against xenobiotics in the gills, thus restricting the transport of toxic substances to other tissues [34]. One member of this family is the P-glycorpotein, which is a highly conserved protein in vertebrate and invertebrate organisms and which is formed by two nucleotide-binding domains (NBD) and two transmembrane (TMB) domains. Moreover, Pgp has two drug binding sites which act as a substrate and which restrict the quantity of drugs incorporated into the cells through ATPasa activity [35]. One of the substrates with the greatest affinity for Pgp are the avermectins, which are used for controlling parasites, among which are nematodes and copepods, by acting as a barrier to the nervous system [3,4,36].
The overuse of xenobiotics for controlling infestations of salmon lice without an adequate control and management system has generated an increased resistance in natural populations. This study characterized the full cDNA of P-glycoprotein in C. rogercresseyi, evidencing a coding sequence of 1,280 amino acids that contain two highly conserved TMB and NBD domains [8,35,36]. The phylogenetic analysis showed that Cr-Pgp had an identity of 76.7% with its homologue describe in L. salmonis [24] and 77.6% of identity with SL-PGY1 describe by Heumann [17]. Moreover, this gene showed a high homology with sequences described for Homo sapiens and Drosophila melanogaster, confirming Cr-Pgp as a member belonging to the ABC transporter family.
In cell lines of mammals, the accumulation of xenobiotic agents has been observed to increase chemical resistance, which is associated with changes in the expression of Pgp [6]. As observed in nematodes treated with avermectins, Pgp increased its transcripts levels and conferred resistance, making treatment with avermectins an inefficient method [3,7]. In L. Salmonis [17,25] and C. rogercresseyi [21], adult copepods exposed to EMB evidences an increase of their Pgp transcriptional activity that in turn affect the efficiency of EMB as chemical antiparasite.
The metabolism of pyrethroids in many species has been associated to overexpression of cytochrome enzyme P450 [36][37][38]. In Helicoverpa armigera at least five CYP genes that encode a P450 enzyme were overexpressed in a resistance strain, suggesting that multiple P450 enzymes are involved in deltamethrin resistance [19]. Besides, an increased sensibility to deltamethrin was observed in L. salmonis after pre-treatment with piperonyl butoxide (PBO), which is a P450 inhibitor, suggesting the role of these enzyme to pyrethroid metabolism in copepods [39]. Bariami et al. [12] in Aedes aegypti identified twenty genes from the P450 superfamily which were overexpressed in resistant strains, also the authors observed an increased level of transcripts of ABC transporter gene, which is orthologous to ABCB4 describe for Anopheles gambiae [39]. These results indicate that ABC transport proteins could be involved in pyrethroid detoxification process. Our results evidenced an overexpression of Cr-Pgp and P450 genes in sea louse exposed to 2 ppb of deltamethrin, suggesting a relation between both genes and a potential resistance to pyrethroid in C. rogercresseyi.   However, these results are different to some previous investigations. In several studies in mouse cell lines, between Pgp and various pesticides demonstrate a low interaction among pyrethroids and Pgp protein [40]. Furthermore, in the parasite Rhipocephalus microplus exposed to ABC transporter inhibitors was observed higher mortality in individuals treated with iveromectin (averomectin), but not in parasites treated with cypermethrin (pyrethroid) [41]. This could indicate an absence of pyrethroid efflux mediated by Pgp in these studied species.
Studies of xenobiotic resistance in salmon lice and the evaluation of Pgp expression has only been carried out in adult individuals exposed to EMB, showing a difference in expression levels between males and females [19]. Analysis from the current study shows that female specimens of C. rogercresseyi presented higher levels of Pgp than males, which is contrary to the information reported for L. salmonis. Additionally, Igboeli [18] observed that the increased expression of Pgp observed in males occurred at an earlier stage than in females, granting males a greater resistance to EMB. From this, it is possible to predict that the greater expression of Pgp observed in females of C. rogercresseyi in the current study could be linked to a greater resistance. This is consistent with the results obtained from Cr-Pgp expression in samples exposed to deltamethrin, where females present higher expression levels in relation to males. In regards to Pgp expression, there are no reports for the earliest developmental stages. It is known that when copepodids are exposed to EMB, development is interrupted, and in the chalimus stage, maturation is delayed [22]. Through expression analysis, it is inferred that copepodids of the analyzed population would be more susceptible to EMB given that the expression Cr-Pgp transcripts in this stage were lower than in the chalimus stage and adult females of C. rogercresseyi. An overregulation of chalimus Cr-Pgp was observed in comparison with expression in copepodids and males, whose expression patterns were similar to those observed in pre-adults and female adults of L. salmonis [19].
The expression and functionality of Pgp could be influenced by the presence of molecular polymorphisms, for instance SNPs within the sequence, which could cause differences in the efficiency of the xenobiotic in different individuals [42,43] evaluated the response of Pgp in humans to changes caused by a SNP (C/T) located in position 3,435, which did not alter the protein but showed an overexpression of the gene in homozygote individuals [42]. In females of Onchocerca volvulus, three SNPs were identified which affected the functionality of Pgp [43]. In reference to the characterization of SL-PGY1 in L. salmonis, no SNPs were reported [17]. However, this study reports 11 SNPs mutations as validated through HRMA. Two of these generated nonsynonymous changes in Met/Leu and Val/Ile. Future studies will evaluate the association of these polymorphisms with resistance or susceptibility to chemotherapeutic agents in the salmon louse C. rogercresseyi [44,45] The present study is the first to describe the mRNA of P-glycoprotein for C. rogercresseyi and simultaneously to measure its expression in adults expose to pyrethroid, and during the larval and adult stages of C. rogercresseyi. The results showed a relation between Cr-Pgp expression and the detoxifying gene Cr-P540, suggesting a role of Cr-Pgp in pyrethroid metabolism.