Markers for monitoring hydrogen peroxide resistance in the salmon louse, Lepeophtheirus salmonis CURRENT

Background: Hydrogen peroxide (H 2 O 2 ) is one of the delousing agents used to control sea lice infestations in salmonid aquaculture. However, some Lepeophtheirus salmonis populations have developed resistance towards H 2 O 2 . An increased gene expression and activity of catalase, an enzyme that breaks down H 2 O 2 , have been detected in resistant lice, being therefore introduced as a resistance marker in the salmon industry. In the present study the aim was to validate the use of catalase expression as a marker and to identify new markers related to H 2 O 2 resistance in L. salmonis . Methods: A sensitive and an H 2 O 2 resistant laboratory strain (P0 generation, not exposed to H 2 O 2 for several years) were batch crossed to generate a cohort with a wide range of H 2 O 2 sensitivities (F2 generation). F2 adult females were then exposed to H 2 O 2 to separate sensitive and resistant individuals. Those F2 lice, the P0 lice and field-collected resistant lice (exposed to H 2 O 2 in the field) were used in an RNA sequencing study. Results: Catalase was up-regulated in resistant lice exposed to H 2 O 2 compared to sensitive lice. This was, however, not the case for unexposed resistant P0 lice. Several other genes were found differentially expressed between sensitive and resistant lice, but most of them seemed to be related to H 2 O 2 exposure. However, five genes were consistently up- or down- regulated in the resistant lice independent of exposure history. The up-regulated genes were: one gene in the DNA polymerase family, one gene encoding a Nesprin-like protein and an unannotated gene encoding a small protein. The down-regulated genes encoded endoplasmic reticulum resident protein 29 and an aquaporin ( Glp1_v2 ). Conclusions: Catalase expression seems to be induced by H 2 O 2 exposure, since it was not up-regulated in unexposed resistant lice. This may pose a challenge for its use as a resistance marker. The five new genes associated with resistance are put forward as potential good, complementary markers. The most promising was Glp1_v2 , an

being directly involved in the resistance mechanism.

Background
The salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) is one of the most important parasitic problems in the Northern hemisphere salmonid aquaculture [1,2]. It also represents a real hazard for wild salmonids [3]. Control of the parasite has historically been based on anti-lice chemicals. However, overuse, in order to keep the louse numbers below the maximum permitted levels in Norway, has led to the development of reduced sensitivity towards most of the available chemical treatments [1,2]. Alternative mechanical and preventive methods have been developed to avoid this problem [3]. Currently, salmon lice control in Norway is based on a combination of preventive, mechanical and chemical delousing methods, as well as extensive monitoring of lice infestation and lice sensitivity to anti-lice treatments [2,4,5,6].
Hydrogen peroxide (H 2 O 2 ) is one of the anti-lice compounds used for controlling salmon lice infestations [7]. It was used between 1993 and 1997 in Norway as a delousing agent, but new chemicals showing better efficacy and with safer margin for fish and farm personnel replaced it. In 2009, H 2 O 2 was reintroduced in the Norwegian salmonid farming industry [8], when reduced efficacy of other chemical treatments was identified [1]. H 2 O 2 is also used in Norwegian aquaculture for treating the amoebic gill disease caused by Paramoeba perurans [9]. As a result, there has been a large increase in the use of this compound in recent years [10] and in 2015, reports on reduced sensitivity towards H 2 O 2 in salmon lice were published [11]. Bioassays on parasites collected in the field, as well as on their descendants, showed a considerable increase in the EC 50 values (the compound concentration affecting 50% of the parasites), confirming higher tolerance to H 2 O 2 in these cohorts compared to parasites from a susceptible laboratory reared strain [11]. H 2 O 2 resistance in salmon lice is an important issue not only in Norway, but also in other salmon producing countries such as Scotland [12].
In biological systems, H 2 O 2 is a naturally occurring reactive oxygen species molecule with cytotoxic effects. It has an important function as a signalling molecule that affects a variety of processes, e.g. immune responses [13]. Several enzymes are involved in the production and regulation of endogenous H 2 O 2 . Therefore, it was not surprising to discover that catalase was involved in the mechanism providing protection to the salmon lice against H 2 O 2 exposure, as this enzyme catalyses the breakdown of H 2 O 2 to H 2 O and O 2 . It was shown that resistant salmon lice had higher catalase gene expression and catalase enzymatic activity compared to sensitive lice [14]. The expression level of catalase was therefore introduced as a H 2 O 2 resistance marker in the salmon industry [15]. An accurate time-space monitoring of the sensitivity level of salmon lice to H 2 O 2 is necessary in order to apply correct control measures. In addition, to know beforehand if the parasites are resistant is highly beneficial in order to avoid the economical, fish welfare and environmental costs of an unsuccessful treatment. Molecular methods have been demonstrated as a powerful tool for monitoring the sensitivity of sea lice to chemicals [16,17], hence the importance of improving and developing such a tool for all anti-lice compounds.
In addition to the catalase enzyme, it would be expected that the lice possess additional mechanisms to protect themselves against high levels of H 2 O 2 [12]. RNA sequencing (RNAseq) is a powerful tool to compare gene expression (as number of transcripts) between selected groups, for all genes simultaneously. This allows for the identification of genes potentially associated with such mechanisms as resistance.
The aims of the present study were to 1) validate the use of the commercially available H 2 O 2 resistance marker (catalase expression), 2) to possibly identify new markers, and 3) to use the annotation of those potential new markers to put forward new hypotheses on the resistance mechanism for H 2 O 2 in salmon lice.

Salmon louse strains
Two well-characterized laboratory L. salmonis strains were used in this study: Ls A, sensitive to all anti-salmon lice compounds used in Norway (tested by bioassays), and Ls V, resistant to azamethiphos, deltamethrin, emamectin benzoate and hydrogen peroxide (field reports and bioassays). Ls A was a strain originally collected on a fish farm in the Northern part of Norway in 2011. Ls V was collected in October 2013, from a fish farm in Mid-Norway with high anti-louse treatment pressure and reported diminished H 2 O 2 treatment efficacy. A total of 14 anti-louse chemical treatments were performed from August 2012 to September 2013 in that farm: six H 2 O 2 treatments (up until one month before the lice collection), six combined treatments with deltamethrin and azamethiphos, one treatment with diflubenzuron and one with emamectin benzoate. The Ls V-2013 samples referred to in the current study were the original field samples of this strain. Ls A and Ls V strains were reared in continuous cultures at the research facilities of Solbergstrand (The Norwegian Institute for Water Research, NIVA, Drøbak, Norway), as described by Hamre et al. [18].
Both strains were maintained without any selection by medicinal compounds.

Crossing experiment and bioassays
In order to obtain lice samples from the same generation and with a range of H 2 O 2 sensitivities, a batch crossing experiment was designed. The experiment was performed as described by Bakke et al. [19] in 2015. Briefly, two Atlantic salmon (one fish per tank) were infested with approximately 50 Ls A copepodids each and another two fish (one fish per tank) with the same number of Ls V copepodids to produce the parental generation (P0). All salmon lice were collected from all fish when the lice were in the pre-adult II stage, before mating occurred. Then 10 pre-adult II Ls A females and 10 pre-adult II Ls V males from the P0 generation were distributed equally on 2 fish kept in individual tanks, to produce the F1 generation of family group 1. The same procedure was used to produce the F1 generation of family group 2, only with opposite gender from each strain, i.e. females from Ls V and males from Ls A. All P0 lice from both family groups were preserved in RNAlater (Sigma, MO, USA) after removal of the egg strings which were set aside to hatch. After ~ 24 h at room-temperature, the preserved samples were stored at − 80 °C. Four fish were infested with copepodids from the F1 generation, two fish with family group 1 and two fish with family group 2. The lice developed to the adult stage, mated, and egg strings for the F2 generation were collected. Approximately 500 copepodids from each of the family groups 1 and 2 (F2) were used for infestation of eight Atlantic salmon for each family group, with the two family groups separated in different tanks.
F2 parasites were selected for sensitivity towards H 2 O 2 (Interox Paramove 50, H 2 O 2 50%, w/w, Solvay 6 Chemicals, Belgium) when they reached the adult stage. The selection was performed in vitro using two-dose bioassays at the Faculty of Veterinary Medicine, NMBU (University of Life Sciences, Oslo, Norway), starting within 6 hours after sampling. All exposures were done in 1 L glass bottles held at 10-12 °C with constant aeration. The females were exposed to either 600 or 1800 ppm H 2 O 2 for 30 min and the results were recorded immediately following exposure [11]. Control groups not exposed to H 2 O 2 were included to check the general performance of the parasites. Parasites affected/immobilized at the lowest H 2 O 2 concentration were considered sensitive, whereas parasites that were not visibly affected at the highest concentration were considered resistant. Lice were classified as affected when they were unable to attach to the container wall (lice could show weak swimming pattern, be partially or completely immobilized at the bottom of the container or float at the surface). Immediately after exposure and recording of the immobilization rate, lice were fixed in RNAlater and kept at − 80 °C following ~ 24 h at room-temperature. Results were expressed as number and percentages of affected lice. A Chi-square test was used to test differences between family groups (statistically significance was assumed when p < 0.05).   [22]. Unmapped reads were filtered out using SAMtools v1.4 [23].
Gene annotation files in GTF format were generated for each parasite and then merged using Cufflinks v2.2.1. [24]. Counts of fragments aligning to each transcript were calculated using  selection events: three on-fish and three off-fish (Table 2). Selection on generation 3 (F3) could not be performed due to low lice numbers. The on-and off-fish selections allowed for two exposure events during the louse lifespan.
For the on-fish selection, fish infested with lice (mostly in the pre-adult I stage) were exposed to 1500 ppm H 2 O 2 for 15-20 min (recommended concentration and exposure time for bath treatments) in a plastic container at 8.5-11 °C and under constant aeration (see Table 2 for details). After treatment, fish were transferred to a recovery container with fresh seawater for 1 h, whereupon they were transferred back to their original tanks. Lice found in the treatment and recovery containers were discarded. The lice remaining on the fish were allowed to develop until females had reached the pre-adult II stage and then selected with H 2 O 2 off-fish. The off-fish method allowed for selection of lice at higher H 2 O 2 concentrations with good re-attachment to fish of the unaffected lice (90-100%).
Briefly, lice were removed from anesthetized fish and transferred to 1 L glass bottles (25-50 lice per bottle), where they were exposed to 2000 or 2500 ppm H 2 O 2 for 30 min (see Table 2 for details). The water was gently mixed every 10 min. Exposures were performed within 4 hours after sampling.
Immediately after exposure, the condition of each louse was recorded. The bottles were emptied and lice attached to the bottle walls were considered unaffected. The bottles were re-filled with 1 L of fresh seawater with constant aeration and lice were left for 1 h -1 h 15 min, after which unaffected parasites were manually put back on the fish (5-10 males and 5-10 females per fish) where they developed to adults and produced eggs for the next generation. Affected lice were discarded. Adult females could not be selected with H 2 O 2 because they were not able to re-attach properly to fish after exposure.
Adult males and females from the F4 generation were removed from anesthetized fish and the egg strings were collected for hatching (F5). Adult F4 females were divided into two groups: One group was immediately fixed in RNAlater, and the other group was exposed to 1000 ppm H 2 O 2 for 30 min (at 10 °C) prior to fixation in RNAlater. The sensitivity to H 2 O 2 (EC 50 ) was determined on the fifth generation (F5). Pre-adult II males and females, and young adult males were used to run a six-dose H 2 O 2 bioassay (see Table 3 for details; 2019 bioassay). A six-dose exploratory H 2 O 2 bioassay was performed before the selection as a reference (Table 3; 2017 bioassay). Both bioassay data were modelled using probit modelling in JMP software, and EC 50 values with 90% confidence intervals were calculated separately for males and females. Chi-square analysis was used to test differences before and after the H 2 O 2 selection (using the number of affected/unaffected lice at each concentration).  Table 3). Table 3 Bioassay data for pre-adult II (males and females) and young adult males exposed to H 2 O 2 for 30 min. Glp1_v2. An elevated expression of catalase has already been associated with resistance towards hydrogen peroxide in males and females preadult stages and in adult males [14],  Ct reference the average of the elongation factor 1-alpha and prohibitin-2 genes (see Table 4 for primer details). Fold change in gene expression was calculated according to the 2^-(∆∆Ct) method, using the Ct values of the corresponding control groups as calibrator sample.

RNAseq expression analysis
RNAseq gene expression analysis (DESeq2) showed that the groups Ls V-2013 and Ls F2-R each had more than 2000 genes differentially regulated compared to the corresponding sensitive groups, Ls A-2013 and Ls F2-S (Fig. 1). The Ls V-P0 lice had less than 150 genes differentially regulated compared to Ls A-P0.  [27], possibly also sooner, although this has not been studied. Several putative methyltransferases and transcription factors (typically involved in gene transcription activation or repression) were found differentially expressed in our RNAseq study (data not shown), supporting the induction theory.

Catalase expression
The catalase gene was previously found differentially expressed in H 2 O 2 -sensitive and -resistant lice [14] and its expression level has been introduced as a H 2 O 2 resistance marker in the salmon industry [15]. The present RNAseq study sought to validate the use of catalase expression as a resistance marker in adult females, as this developmental stage was not included in a previous study [14]. There were significantly higher numbers of catalase transcripts in resistant lice exposed to H 2 O 2 (Ls V-2013 and Ls F2-R) than in sensitive lice (   5). However, the inter-individual variation in the exposed group, was smaller than in the unexposed one. A similar trend is observed among sensitive lice enrolled in the RNAseq study: Ls A-2013 and Ls A-P0 (unexposed) vs F2-S (exposed) ( Table 6 and Fig. 2).
H 2 O 2 resistance has been demonstrated to be hereditary [11,14]. The heritable factor may thus be the ability to quickly induce catalase expression. (Ls V-2013 and Ls F2-R) shared 790 differentially expressed genes (Fig. 1). This supports the hypothesis that H 2 O 2 exposure can induce the expression of several genes, even within a time-span of 30 minutes. The complete list of genes shared across two or all three groups is presented in Additional files 2.
Only five genes (three up-regulated and two down-regulated in resistant lice) were differentially expressed in all three groups (Ls V-2013, Ls V-P0 and Ls F2-R) (Figs. 1 and 2), thus irrespective of H 2 O 2 exposure. Table 6 shows the gene expression and annotation data for those genes. The log2 fold change ranged from ~|0.2| to ~|3|. The three genes consistently up-regulated in resistant lice encoded a DNA polymerase (possibly delta subunit 3), a Nesprin-like protein and an unannotated small protein (named NA; 77 aa long). DNA polymerase is an enzyme that synthesize DNA from deoxyribonucleotides, and the delta subunit 3 plays a role in high fidelity genome replication. The protein identified as Nesprin-like contained a KASH domain and a spectrin repeat (spectrin/alphaactinin). It probably belongs to the Nesprin-1 or -2 type, actin-binding proteins involved in the maintenance of nuclear organization and structural integrity. The NA protein might be a mini-protein with regulatory functions. A large amount of mini-and micro-proteins (small proteins usually < 100 aa long) acting as negative or positive regulators, have being identified in unicellular organisms, plants and animals [28,29]. For example, some small proteins sequester their targets into non-functional complexes, others attract chromatin repressor proteins, or others interact with ion channels compromising their transport capacity.
The two genes down-regulated in all resistant lice were the genes encoding endoplasmic reticulum resident protein 29 (ERP29) and an aquaporin protein (Glp1_v2). ERP29 plays an important role in the processing of secretory proteins within the endoplasmic reticulum. Aquaporins are protein channels that facilitate the rapid transport of water, other small solutes such as H 2 O 2 and gasses [22,30,31,32,33,34].
Glp3_v2 was expressed mostly in nauplius II stage. Table 6 shows the gene expression data for several aquaporins in our study. There were no statistically significant differences in the expression of It has been demonstrated that certain aquaglyceroporins and unorthodox aquaporins are able to transport H 2 O 2 through cell membranes in several organisms [32,33].
Glps have an open pore configuration in L. salmonis [22], allowing bigger molecules than water, like urea and glycerol, to pass through the channel. Miller et al. [32], found that one aquaglyceroporin (AQP3) and one unorthodox aquaporin (AQP8) transported H 2 O 2 through mammalian cell membranes. However, the classical aquaporin AQP1, did not transport H  (Table 6). As in the case of Glps, Stavang et al. [22] also found an open pore configuration in the 3D modelling of Aqp12L2.
Several authors have reported the role of aquaporins as drug transporters in other parasites, as well as a link between aquaporins and drug resistance [35]. Faghiri and Skelly [36], showed the presence of a putative aquaglyceroporin (SmAQP) in the tegument of the parasitic worm Schistosoma mansoni.
It was proven that SmAQP can transport water and an anti-parasitic compound (potassium antimonyl tartrate) across the parasite tegument. In addition, parasites with reduced levels of SmAQP exhibited a greater resistance to the anti-parasitic agent. In Trypanosomatid parasites, like Leishmania or Trypanosoma spp., certain aquaporins transport trivalent metalloids (SbIII and AsIII) through the parasite membranes [37]. The aquaglyceroporin LmAQP1 transports SbIII in Leishmania spp [38].
Drug resistant parasites showed down-regulation of the LmAQP1 gene [39], and RNA levels correlated with drug concentration. Resistance to melarsoprol and pentamidine is common among African trypanosomes [40]. The authors found that the loss of function of an aquaglyceroporin, AQP2, was linked to drug resistance.
Studies have shown that the amount of functional proteins can be related to the amount of RNA transcripts, but also to the activation state or degradation rate of the proteins. For example, a mitogen activated protein kinase 2 (MPK2) stabilizes LmAQP1 protein by phosphorylation in Leishmania major [41], and dephosphorylation made LmAQP1 more susceptible to degradation.
Altered AQP1 and MPK2 (by site-directed mutagenesis) reduced the drug uptake and drug sensitivity.
Catalase activity can also be regulated by reversible phosphorylation via kinase enzymes by increasing the affinity of the enzyme for H 2 O 2 [42]. In our L. salmonis RNAseq study, we found four putative mitogen activated protein kinases differentially expressed in H 2 O 2 sensitive and resistant lice (data not shown), indicating that drug sensitivity might be linked to regulation of gene expression, but also to the amount and functionality of the proteins.  and compared with Ls V-P0 lice (not exposed to H 2 O 2 for two years). F4 lice were exposed twice to H 2 O 2 , as pre-adult I and pre-adult II ( Table 2). When the females became adults, they were either

Ethics approval and consent to participate
The use of fish for salmon lice cultivation was approved by the Norwegian Food Safety Authorities     qPCR study for catalase expression in the original laboratory Ls A strain (sensitive to H2O2, adult females). Ls A 0 ppm: Unexposed lice (N=5; white rectangles); Ls A 600 ppm: lice exposed to 600 ppm H2O2 for 30 min (N=5; grey rectangles; unaffected after the exposure). Solid lines represent the mean in each group. Data shown as fold change (log2^-(∆∆Ct)) referred to Ls A 0 ppm lice (calibrator sample). Statistical analysis was not performed due to the low sample size in each group.