Evolutionary history and diversification of duplicated fatty-acyl elongase genes of Atlantic salmon (Salmo salar)

Background: The Atlantic salmon, Salmo salar L., is a prominent member of the Salmonidae family, and has been the focus of intense research because of its environmental and economic significance as an iconic sporting species and its global importance as an aquaculture species. Furthermore, salmonids constitute ideal organisms for the study of evolution by gene duplication as they are pseudotetraploid descendants of a common ancestor whose genome was duplicated some 25 to 100 million years ago. Whole-genome duplication is considered a major evolutionary force capable of creating vast amounts of new genetic material for evolution to act upon, promoting speciation by acquisition of new traits. Recently, large-scale comparison of paralogous genes in Atlantic salmon suggested that asymmetrical selection was acting on a significant proportion of them. However, to elucidate the physiological consequences of gene and genome duplications, studies integrating molecular evolution and functional biology are crucial. To this end, sequence and molecular analyses were performed on duplicated Elovl5 fatty-acyl elongases of Atlantic salmon, as they are responsible for a rate-limiting reaction in the elongation process of long-chain polyunsaturated fatty acids (LC-PUFA), critical components of all vertebrates. The aim of the research presented here was to investigate the role of gene duplication as an evolutionary process capable of creating genetic novelty, and to identify the potential ecological and physiological implications. Results: Linkage analyses indicated that both fatty-acyl elongases segregated independently and located elovl5 duplicates on different linkage groups. Genetic mapping using microsatellites identified in each elovl5 locus assigned elovl5a and elovl5b to chromosomes ssa28 and ssa13, respectively. In silico sequence analysis and selection tests indicated that both salmon Elovl5 proteins were subject to purifying selection, in agreement with previous results showing indistinguishable substrate specificities. Gene expression and promoter analysis indicated that Elovl5 duplicates differed in response to dietary lipids and tissue expression profile. Lipid biosynthesis and metabolic gene expression profiling performed in Atlantic salmon SHK-1 cells, suggested that the control of lipid homeostasis in fish is similar to that described in higher vertebrates, and revealed the particular importance of Lxr and Srebp transcription factors (TFs) in the regulation of LC-PUFA biosynthetic enzymes. Sequence comparison of upstream promoter regions of elovl5 genes showed intense differences between duplicates. Promoter functional analysis by co-transfection and transcription factor transactivation showed that both elovl5 duplicates were upregulated by Srebp overexpression. However, elovl5b exhibited a higher response and its promoter contained a duplication of a region containing response elements for Srebp and NF-Y cofactors. Furthermore, these studies indicated an Lxr/Rxr dependant response of elovl5a, which was not observed in elovl5b. Analysis of the genomic sequences of elovl5 duplicates by comparison to various sequence databases showed an asymmetrical distribution of transposable elements (TEs) in both introns and promoter regions. Further comparison to introns of the single elovl5 gene in pike indicated much higher TE distribution in salmon genes compared to the pike. Conclusions: Although not conclusive, the most parsimonious origin for the salmon elovl5 duplicates is that they are derived from a WGD event. This conclusion is also supported by the close similarity of two elovl5 paralogs in the recently available rainbow trout genome. Regardless of their origin, Atlantic salmon elovl5 genes have been efficiently retained in the genome under strong functional constraints indicating a physiological requirement for both enzymes to be functionally active. In contrast, upstream promoter regions have strongly diverged from one another, indicating a relaxation of purifying selection following the duplication event. This divergence of cis-regulatory regions has resulted in regulatory diversification of the elovl5 duplicates and regulatory neofunctionalisation of elovl5a, which displayed a novel Lxr/Rxr-dependant response not described in sister or other vertebrate lineages. Promoter analysis indicated that the observed elovl5 differential response to dietary variation could be partly attributed to varying transcriptional regulation driven by lipid-modulated TFs. The distribution of TEs in elvol5 genes of Atlantic salmon shows a clear increase in TE mobilisation after the divergence of esocids and salmonids. This must have occurred after the elongase duplication and thus the salmonid WGD event and contributes to the observed regulatory divergence of elovl5 paralogs

Genome-wide survey of cytochrome P450 genes in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837)

Background The salmon louse (Lepeophtheirus salmonis) infests farmed and wild salmonid fishes, causing considerable economic damage to the salmon farming industry. Infestations of farmed salmon are controlled using a combination of non-medicinal approaches and veterinary drug treatments. While L. salmonis has developed resistance to most available salmon delousing agents, relatively little is known about the molecular mechanisms involved. Members of the cytochrome P450 (CYP) superfamily are typically monooxygenases, some of which are involved in the biosynthesis and metabolism of endogenous compounds, while others have central roles in the detoxification of xenobiotics. In terrestrial arthropods, insecticide resistance can be based on the enhanced expression of CYPs. The reported research aimed to characterise the CYP superfamily in L. salmonis and assess its potential roles in drug resistance. Methods Lepeophtheirus salmonis CYPs were identified by homology searches of the genome and transcriptome of the parasite. CYP transcript abundance in drug susceptible and multi-resistant L. salmonis was assessed by quantitative reverse transcription PCR, taking into account both constitutive expression and expression in parasites exposed to sublethal levels of salmon delousing agents, ecdysteroids and environmental chemicals. Results The above strategy led to the identification of 25 CYP genes/pseudogenes in L. salmonis, making its CYP superfamily the most compact characterised for any arthropod to date. Lepeophtheirus salmonis possesses homologues of a number of arthropod CYP genes with roles in ecdysteroid metabolism, such as the fruit fly genes disembodied, shadow, shade, spook and Cyp18a1. CYP transcript expression did not differ between one drug susceptible and one multi-resistant strain of L. salmonis. Exposure of L. salmonis to emamectin benzoate or deltamethrin caused the transcriptional upregulation of certain CYPs. In contrast, neither ecdysteroid nor benzo[a]pyrene exposure affected CYP transcription significantly. Conclusions The parasite L. salmonis is demonstrated to possess the most compact CYP superfamily characterised for any arthropod to date. The complement of CYP genes in L. salmonis includes conserved CYP genes involved in ecdysteroid biosynthesis and metabolism, as well as drug-inducible CYP genes. The present study does not provide evidence for a role of CYP genes in the decreased susceptibility of the multiresistant parasite strain studied

Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite causing infections ofwild and farmed Atlantic salmon (Salmo salar L.) in the Northern hemisphere.While L. salmonis control at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved through L. salmonis bioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitable L. salmonis stages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried outwith limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the timepointwhere exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individual L. salmonis by determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains of L. salmonis differing in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50 showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistant L. salmonis laboratory strains. Statement of relevance: Infections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment. The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms,where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to showa response after drug treatment has started, thus providing a variable characterizing the drug susceptibility phenotype of individual parasites. Accordingly, the bioassay approach presented here will be useful for studies aiming at unravelling the genetic determinants of drug resistance

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis)

Background Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage‐gated sodium channels (Nav1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterised Nav1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. Results Genome scans identified three L. salmonis Nav1 homologues, LsNav1.1, LsNav1.2 and LsNav1.3. Arthropod kdr mutations map to specific Nav1 regions within domains DI‐III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT‐PCR and sequenced in deltamethrin‐susceptible and deltamethrin‐resistant L. salmonis. While LsNav1.1 and LsNav1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav1.3 showed a non‐synonymous mutation in S5 of DII occurring in deltamethrin‐resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid‐resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid‐susceptible strains. Conclusions The present study indicates that a kdr‐mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis

Investigation of deltamethrin resistance in salmon lice (Lepeophtheirus salmonis) provides no evidence for roles of mutations in voltage-gated sodium channels

BACKGROUND The pyrethroid deltamethrin is used to treat infestations of farmed salmon by parasitic salmon lice, Lepeophtheirus salmonis (Krøyer). However, the efficacy of deltamethrin for salmon delousing is threatened by resistance development. In terrestrial arthropods, knockdown resistance (kdr) mutations of the voltage‐gated sodium channel (Nav), the molecular target for pyrethroids, can cause deltamethrin resistance. A putative kdr mutation of an L. salmonis sodium channel homologue (LsNav1.3 I936V) has previously been identified. At the same time, deltamethrin resistance of L. salmonis has been shown to be inherited maternally and to be associated with mitochondrial DNA (mtDNA) mutations. The present study assessed potential roles of the above putative kdr mutation as a determinant of deltamethrin resistance in laboratory strains and field populations of L. salmonis. RESULTS The deltamethrin resistant L. salmonis strain IoA‐02 expresses the LsNav1.3 I936V mutation but was susceptible to the non‐ester pyrethroid etofenprox, a compound against which pyrethroid resistant arthropods are usually cross‐resistant if resistance is caused by Nav mutations. In a family derived from a cross between an IoA‐02 male and a drug‐susceptible female lacking the kdr mutation, deltamethrin resistance was not associated with the genotype at the LsNav1.3 locus (p>0.05). Similarly, in Scottish field populations of L. salmonis, LsNav1.3 I936V showed no association with deltamethrin resistance. In contrast, genotypes at the mtDNA loci A14013G and A9030G were significantly associated with deltamethrin resistance (P less than 0.001). CONCLUSION In the studied L. salmonis isolates, deltamethrin resistance was unrelated to the LsNav1.3 I936V mutation, but showed close association with mtDNA mutations

Maternal inheritance of deltamethrin resistance in the salmon louse Lepeophtheirus salmonis (Krøyer) is associated with unique mtDNA haplotypes

Parasitic infections by the salmon louse, Lepeophtheirus salmonis (Krøyer), cause huge economic damage in salmon farming in the northern hemisphere, with combined treatment costs and production losses in 2014 having been estimated at US$ 350 million for Norway (annual production 1.25 million tonnes). The control of L. salmonis relies significantly on medicinal treatments, supplemented by non-pharmacological approaches. However, efficacy losses have been reported for several delousing agents, including the pyrethroid deltamethrin. The aim of the present study was to analyse the genetic basis of deltamethrin resistance in L. salmonis. Deltamethrin median effective concentrations (EC50) were 0.28 μg L-1 in the drug susceptible L. salmonis strain IoA-00 and 40.1 μg L-1 in the pyrethroid resistant strain IoA-02. IoA-00 and IoA-02 were crossed to produce families spanning one parental and three filial generations (P0, F1-F3). In three families derived from P0 crosses between an IoA-00 sire and an IoA-02 dam, 98.8% of F2 parasites (n = 173) were resistant, i.e. remained unaffected after exposure to 2.0 μg L-1 deltamethrin. F3 parasites from these crosses showed a deltamethrin EC50 of 9.66 μg L-1. In two families of the inverse orientation at P0 (IoA-02 sire x IoA-00 dam), 16.7% of F2 parasites were resistant (n = 84), while the deltamethrin EC50 in F3 animals was 0.26 μg L-1. The results revealed a predominantly maternal inheritance of deltamethrin resistance. The 15,947-nt mitochondrial genome was sequenced and compared among six unrelated L. salmonis strains and parasites sampled from wild salmon in 2010. IoA-02 and three further deltamethrin resistant strains, established from isolates originating from different regions of Scotland, showed almost identical mitochondrial haplotypes. In contrast, the mitochondrial genome was variable among susceptible strains and L. salmonis from wild hosts. Deltamethrin caused toxicity and depletion of whole body ATP levels in IoA-00 but not IoA-02 parasites. The maternal inheritance of deltamethrin resistance and its association with mitochondrial haplotypes suggests that pyrethroid toxicity in L. salmonis may involve molecular targets encoded by mitochondrial genes

A survey of the ATP-binding cassette (ABC) gene superfamily in the salmon louse (Lepeophtheirus salmonis)

Salmon lice,Lepeophtheirus salmonis(Krøyer, 1837), are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon,Salmo salarLinnaeus, 1758. The control ofL.salmonisat fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which inL.salmonisis documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC) gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters inL.salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes inL.salmonisfor which, ABC superfamily members were identified through homology searching of theL.salmonisgenome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq) of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters,i.e. subfamilies B (4 sequences), C (11) and G (2). The results suggest that the ABC gene family ofL.salmonispossesses fewer members than recorded for other arthropods. The present survey of theL.salmonisABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance

Phylogenetic analysis of ABCC transporters of <i>H</i>. <i>sapiens</i> and six invertebrate species.

<p>Full-length coding regions were aligned using MUSCLE and subjected to a maximum likelihood analysis using RAxML. For further explanations see the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137394#pone.0137394.g002" target="_blank">Fig 2</a>.</p

Summary of 33 ABC proteins identified in <i>L</i>. <i>salmonis</i>.

<p>n/a, not applicable; AA, amino acid; TM, transmembrane domain; NDB, nucleotide binding domain</p><p>¥ Predicted protein (Database LSalAtl2EbiPred6; <a href="http://sealouse.imr.no/" target="_blank">http://sealouse.imr.no/</a>).</p><p>† Partial sequence only.</p><p>* Alternative splicing forms exist.</p><p>Summary of 33 ABC proteins identified in <i>L</i>. <i>salmonis</i>.</p

Phylogenetic analysis of ABCB full transporters of <i>H</i>. <i>sapiens</i> and six invertebrate species.

<p>Full-length coding regions were aligned using MUSCLE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137394#pone.0137394.ref042" target="_blank">42</a>] and subjected to a maximum likelihood analysis using RAxML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137394#pone.0137394.ref044" target="_blank">44</a>]. Colour diamonds are as follows: blue, <i>C</i>. <i>elegans</i>; yellow, <i>H</i>. <i>sapiens</i>; grey, <i>T</i>. <i>castaneum</i>; green, <i>D</i>. <i>melanogaster</i>; purple, <i>D</i>. <i>pulex</i>; orange, <i>T</i>. <i>urticae</i> and red, <i>L</i>. <i>salmonis</i>. The scale bar represents amino-acid substitutions per site. Accession numbers of used sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137394#pone.0137394.s014" target="_blank">S4 Table</a>.</p