Sex-biased gene expression and sequence conservation in Atlantic and Pacific salmon lice (Lepeophtheirus salmonis)

Salmon lice, Lepeophtheirus salmonis (Copepoda: Caligidae), are highly important ectoparasites of farmed and wild salmonids, and cause multi-million dollar losses to the salmon aquaculture industry annually. Salmon lice display extensive sexual dimorphism in ontogeny, morphology, physiology, behavior, and more. Therefore, the identification of transcripts with differential expression between males and females (sex-biased transcripts) may help elucidate the relationship between sexual selection and sexually dimorphic characteristics. Sex-biased transcripts were identified from transcriptome analyses of three L. salmonis populations, including both Atlantic and Pacific subspecies. A total of 35-43 % of all quality-filtered transcripts were sex-biased in L. salmonis, with male-biased transcripts exhibiting higher fold change than female-biased transcripts. For Gene Ontology and functional analyses, a consensus-based approach was used to identify concordantly differentially expressed sex-biased transcripts across the three populations. A total of 127 male-specific transcripts (i.e. those without detectable expression in any female) were identified, and were enriched with reproductive functions (e.g. seminal fluid and male accessory gland proteins). Other sex-biased transcripts involved in morphogenesis, feeding, energy generation, and sensory and immune system development and function were also identified. Interestingly, as observed in model systems, male-biased L. salmonis transcripts were more frequently without annotation compared to female-biased or unbiased transcripts, suggesting higher rates of sequence divergence in male-biased transcripts. Transcriptome differences between male and female L. salmonis described here provide key insights into the molecular mechanisms controlling sexual dimorphism in L. salmonis. This analysis offers targets for parasite control and provides a foundation for further analyses exploring critical topics such as the interaction between sex and drug resistance, sex-specific factors in host-parasite relationships, and reproductive roles within L. salmonis.


Background
Sexual dimorphism describes the phenotypic differences between sexes of the same species. It is ubiquitous across the animal kingdom and is favored through a combination of sexual selection, intersexual competition for resources, and fundamental differences in reproductive roles [1][2][3]. Genes overexpressed in one sex relative to the other are known as sex-biased genes, and include genes expressed in both sexes (but higher in one) or genes expressed in only one sex (sex-specific; reviewed in [4,5]). A large proportion, often greater than 50 %, of genes exhibit sex-biased expression in many species including fruit flies Drosophila spp. [6,7], the nematode Caenorhabitis elegans [8], parasitic flatworms Schistosoma spp. [9,10], the water flea Daphnia pulex [11], the African clawed frog Xenopus laevis [12], the songbirds Taeniopygia guttata and Sylvia communis [13], the olive flounder Paralichthys olivaceus [14], the mouse Mus musculus [15], and humans Homo sapiens [16,17]. This trend is largely driven by expression differences in the gonad. As such, transcriptome profiling is a highly useful approach to understand the mechanisms underlying sexual dimorphism and reproduction.
Crustaceans are one of the most diverse animal taxa, comprising more than 850 families with approximately 67,000 species [18,19]. They are ecologically important, serving essential roles in the food chain and primary production in marine ecosystems [20]. Furthermore, crustaceans play important roles in aquaculture as both farmed animals (62 species worth over USD 34.8 billion per year; [21,22]) and as parasites of farmed fish [23]. Most parasitic crustaceans are species within the class Copepoda, which displays a vast array of sexual dimorphism in anatomy, reproductive roles, sensory systems, and host/parasite relationships [23]. One of the most studied parasitic copepod, the salmon louse Lepeophtheirus salmonis, causes more than USD 480 million in losses to the Atlantic Salmon (Salmo salar) aquaculture industry annually [24]. Additionally, drug resistant strains of L. salmonis (and other sea lice species) have emerged globally (reviewed by [25]), threatening the sustainability and productivity of the industry.
Lepeophtheirus salmonis displays sexual dimorphism among several morphological, physiological, and behavioural characters. This phenomenon is observed in the motile parasitic stages of the lice (pre-adult I, II, and adult) although sex-specific differences in cephalothorax size and molt timing are also evident at preceding stages [26]. In addition, males develop faster than females, but they mature at approximately half the size of the adult female [26][27][28]. Sex differences in the morphology of the genital segment, abdomen, and appendages occur in all motile stages [27]. Distinct sex-associated behavioral characteristics related to reproductive success including frequency of host switching [29][30][31], mate location [32], blood feeding [33], and mate-guarding [31,34] have also been reported. Sexually dimorphic physiology is also evident when L. salmonis are exposed to a commonly used antiparasitic compound, emamectin benzoate (EMB). Although EMB-resistance is widespread [25], males consistently show higher tolerance to EMB compared with females, regardless of the overall level of resistance within the population [35][36][37]. However, the molecular mechanisms underpinning sex-specific anatomy, behavior, and physiology in L. salmonis, and copepods in general, remain poorly understood.
The present study investigates sex-biased gene expression in three populations of L. salmonis using newlygenerated transcriptomic data from Pacific Canada L. salmonis as well as a novel analysis of an available published dataset from Atlantic Canada L. salmonis [37]. A consensus-based, meta-analysis approach was used to identify sex-biased transcripts putatively responsible for sexual dimorphism in L. salmonis. Additionally, L. salmonis sequence conservation with related species (UniProt or Conserved Domain Database; e < 10 -10 ) was integrated with sex-biased expression results to investigate sexspecific selective pressure and genomic constraint.

Sex-biased gene expression in L. salmonis
Sex-biased transcripts were identified in three populations of pre-adult II L. salmonis using a 38 K oligonucleotide microarray. Two of the populations were from the Atlantic subspecies L. salmonis salmonis and were collected from separate bay management areas (BMA-2a and BMA-2b) in the Bay of Fundy, New Brunswick [37], and the third was from the Pacific subspecies L. salmonis oncorhynchi [38] collected from the Broughton Archipelago, British Columbia (BC). Eighteen to 21 F1 generation preadult males and females from each population were analyzed in individual microarray hybridizations (total n = 117 individuals and hybridizations). A total of 34.7 -42.7 % of all unique contigs passing quality control (QC) filters were significantly sex-biased (Benjamini-Hochberg multiple test correction; p < 0.01; fold change (FC) ≥ 1.5) in Atlantic and Pacific L. salmonis (Table 1). Including only the transcripts expressed in both sexes, a Principal Component Analysis (PCA) separated male and female samples along the first principal component (PC1; explaining the most variation) in all three populations, representing 50.2, 39.5 and 53.4 % of the transcriptional variation in BMA-2a, BMA-2b, and Pacific lice, respectively (Fig. 1). No consistent differences were observed between the proportions of transcripts overexpressed in males relative to females in each population (Table 1). Sex-biased transcripts for each population, including p-values, fold changes, annotations, and accession identifiers, can be found in Additional file 1.

Sequence conservation in sex-biased transcripts
The L. salmonis contigs used for microarray construction [39] were annotated using BLASTx and RPS-BLAST against SwissProt and Conserved Domain Database (CDD; [40]), respectively [39]. Contigs without annotation (e > 10 -10 ) are marked as unknown in the additional files. The proportion of orphans relative to annotated transcripts in male-biased, female-biased, and unbiased categories was assessed for each population. This approach has been used in other model organisms, for example in flies and nematodes [11,41]. Male-biased transcripts from all three L. salmonis populations had a higher proportion of orphans compared with female-biased and unbiased transcripts (Table 1). Female-biased and unbiased transcripts did not show consistent differences in the proportion of orphans (Table 1). These data suggest lower sequence conservation of male-biased transcripts in L. salmonis.
Consensus of sex-biased transcripts in three populations of L. salmonis To assess the functional impacts of sex-biased expression in L. salmonis, differentially expressed transcripts between sexes from each population were used to generate a consensus list (Fig. 2). A total of 1470 unique transcripts, out of a total of 7889 were shown to be Additional file 2). Using this consensus list (i.e. requiring concordant differential expression being identified in all three populations), 368 transcripts showed male-bias and 461 were female-biased. As expected from the individual population analyses, consensus male-biased transcripts showed a 2.5-fold higher proportion of orphans compared with those showing female-bias (Table 1). Differences in the degree of sex-biased expression, as measured by fold change (FC) also varied between male-and female-biased transcripts. On average, 84.8 % of the transcripts overexpressed in females had low sex-bias (FC ≥ 1.5 and ≤ 4), whereas transcripts overexpressed in males had equal proportions of high and low sex-bias (Table 2). Interestingly, 127 male-biased transcripts were not expressed above background levels in any of the 58 females assayed and therefore are referred to as malespecific. In contrast, only 20 transcripts were femalespecific in the consensus list. Here, fold changes are reported as the range of differential expression between males and females across all populations, unless the transcript was sex-specific, and then it is denoted as such. Fold changes specific to each population for consensus sex-biased transcripts can be found in Additional file 2.
Male-biased transcripts in three populations of L. salmonis The majority of annotated male-biased transcripts had roles in reproduction, for example being accessory gland proteins (Acps) and seminal fluid proteins (SFPs; reviewed by [42]). Transcripts known to regulate proteolysis for reproduction-related functions were highly male-biased in L. salmonis and included 16 proteases and 13 protease inhibitors, 10 of each being male-specific. However, a high degree of variance was observed in the expression of proteolytic transcripts among Pacific males (Fig. 3). To better understand this expression pattern, a transcript similarity assessment using kunitz/ BPTI-like toxin (probe ID: C259R052) showed that 110 transcripts were strongly co-expressed (Pearson's correlation, 0.95 < r < 1.0; Fig. 3). Although Atlantic males showed constitutive expression of these transcripts, Pacific males showed a characteristic "on/off" expression profile, with 10 of 19 individuals showing low, or absence of expression (Fig. 3). This co-expressed transcript list contains numerous representatives from known functional categories of male reproduction including peroxidases, pH regulators, kinases, and transporters, among others ( Table 3). As seminal fluid proteins (SFPs) are only expressed in males [43], transcripts exhibiting male-specific expression are putatively assigned as candidate SFPs in L. salmonis.
Other sex-biased transcripts involved in the nervous system, including their sex-biased expression profiles, are reported in Fig. 4. As salmon lice display sexually dimorphic patterns of mobility (i.e. mate location [32] and frequency of host switching [31]) and responses to neurotoxic drugs [37,44], these transcripts will serve as important markers to better understand sex-related differences in the L. salmonis sensory system.

Annotation of L. salmonis sex-biased orphan contigs
Sex-biased orphan contigs were compared to known sequences using UniProt (e ≤ 10 -5 ) to augment novel transcript discovery in the non-model L. salmonis. Although this threshold is less conservative than that used for the original microarray annotation [39], it has been frequently used in other sea lice transcriptomic studies [47][48][49], being generally acceptable for gene annotation [50]. This method was used for novel transcript discovery only and these newly annotated transcripts were not included in GO analyses. A total of 16 female-biased and References are provided based on the identification of similarly annotated sequences or proteins involved in male reproduction (e.g. spermatogenesis, seminal fluid proteins, testis expression) in related organisms. The degree of male-bias is indicated by a range of fold change (FC) across populations while MS indicates the listed probes showed male-specific expression. Transcripts denoted with a have a signal peptide for secretion (SwissProt) while those with b were annotated using e < 10 -5 due to the absence of homology at higher stringency. Transcripts without annotation are represented by NA (no annotation). Each transcript has a unique contig ID which can be found in Additional file 2 using the probes listed here 12 male-biased transcripts were subsequently annotated (Additional file 2) using this method. This additional annotation did not substantially change observations made on sequence divergence differences in male and femalebiased transcripts, and many of the newly annotated female-biased transcripts had similar functions to those identified in the enrichment analysis described above, including transcription, translation, and cell cycle (Additional file 3). Potential links to reproduction were observed among several of the newly annotated male-biased orphans, including prostasin (male-specific), gamma-glutamyltranspeptidase (FC = 1.7 -3.4), and protein ref(2)P (FC = 1.6 -2.3) ( Table 3). A male-specific transcript, c-factor, was also identified. However, the function of this transcript in L. salmonis remains unknown. Additionally, male-biased transcripts involved in neuromuscular development and function were discovered, including excitatory amino acid transporter 3 (SLC1A1; FC = 2.3 -3), twitchin (FC = 1.6 -5.7), and sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (FC = 1.5 -7.6). Probe identifiers, e-values, bitscores, SwissProt accessions, and descriptions for newly annotated transcripts can be found in Additional file 2.

Discussion
The economically and ecologically important parasite, salmon lice L. salmonis (Copepoda: Caligidae) displays sexual dimorphism in ontogeny, morphology, physiology, and behavior [26][27][28][29][30][31][32][33][34][35]. However, little is known about the molecular mechanisms that control these traits and the possible interactions they may have with chemical response/resistance, host-parasite interactions, and overall population dynamics. Here, sex-biased transcripts were identified within three populations of L. salmonis (including two subspecies; [38]) from the Atlantic and Pacific coasts of Canada. Our observation revealed a large proportion (34.7 -42.7 %) of sex-biased expression (Table 1) consistent with findings in other arthropods [6,11], nematodes [8], amphibians [12], fish [51], birds [13], and mammals [17]. Principal component analysis supported this finding as males and females were separated on PC1 in all populations. These data suggest large differences in baseline gene expression between male and female L. salmonis that could potentially impact parasite control strategies. For example, routine lice counting and staging is the primary management strategy used to inform treatment regimes in aquaculture. However, pre-adult male and female L. salmonis are often grouped into a single category for these counts [52,53]. The basal differences in gene expression reported here and the interactive effects of sex with chemical treatment and resistance described elsewhere [37], suggests a more informative strategy would be to separately count male and female preadult L. salmonis in both farm and laboratory settings whenever possible.
There were no consistent differences between the proportion of male-biased relative to female-biased transcripts in L. salmonis (Table 1), despite observations in other crustaceans like Caligus rogercresseyi, another species of parasitic copepod, and Daphnia pulex which suggested a slight over-representation of male-biased transcripts [11,54]. However, we did find that a larger proportion of malebiased transcripts had higher fold change than those with female-bias. In this study, sex-biased expression was assessed across multiple populations of L. salmonis reared under similar conditions, thus offering a more comprehensive overview of gene expression differences than is obtained in single cohort transcriptome analyses. However the sex-biased transcripts identified here were limited tõ 22,000 unique contig sequences (i.e. transcripts) on the microarray and it was not possible to examine some of the more complex facets of sex-biased expression (i.e. sexspecific alternative transcripts [55]). Additionally, studies using whole-body homogenates offer general patterns of sex-bias but lack the resolution to detect sex-biased expression in individual tissues [7] and control for differences in tissue allometry [56]. Tissue-specific extractions will be an important tool for future studies examining sex-biased gene expression in L. salmonis. Nonetheless, characterization of sex-biased transcripts reported here provides numerous molecular targets putatively underlying sexual dimorphism in L. salmonis, offering important insight for pest management and future drug development for this non-model organism.

Discovery of sex-biased transcripts related to reproduction in L. salmonis
Male-biased transcripts displayed greater sex-bias compared with female-biased transcripts in L. salmonis, similar to findings in D. melanogaster [6]. Among the highly male-biased transcripts, 127 were not expressed above background detection in any of the 58 females assayed in this study. These transcripts represent putative seminal fluid proteins (SFPs) based on their expression profiles (SFPs are only expressed in males; [43]) and annotations (Table 3 and references therein). SFPs are transferred to females during mating, impacting a variety of physiological processes such as sperm storage, egg production, feeding, behavior, and receptivity to remating (reviewed in [42]). Although genes involved in reproduction tend to evolve rapidly [57], many of the functional constituents of seminal fluid are conserved from arthropods to mammals [43,58]. For example, genes involved in proteolysis are essential for sperm transfer, storage, and activity, among other functions [59,60]. A total of 10 proteases and 10 protease-inhibitors were identified as male-specific in this study with several others showing male-bias (Table 3). All protease inhibitors putatively involved in reproduction contained at least one kunitz or serpin domain (CDD accessions: pfam00014 and cd00172, respectively), which are the predominant classes of protease inhibitors in seminal fluid [59]. In turn, proteases such as serine protease persephone and calpain-A catalytic subunit also had relevant annotations to SFPs based on their ability to modulate toll signaling in D. melanogaster [61,62]. The transfer of immunomodulatory and antimicrobial SFPs may aid females in defending against infections that are introduced during mating [60,63]. Many of the L. salmonis proteases assigned as SFPs also contained trypsin-like domains (CDD: cd00190 and smart00020) including prostasin, proclotting enzyme heavy chain, and three additional transcripts without SwissProt annotation (i.e. CDD only). These transcripts represent important targets for understanding the proteolytic events controlling reproduction and mating in L. salmonis.
Female-biased transcripts also had putative reproductive functions, including five with nuclear hormone receptor activity, one of which was female-specific. Recently, a sex-linked SNP in the L. salmonis prohibitin-2 gene with a pattern of female-biased expression was identified in three strains of L. salmonis from Scotland [45]. Similarly, a transcript annotated as prohibitin-2 was female-biased in all three populations of L. salmonis assayed in this study. The prohibitin-2 gene is likely involved in sex determination [45] along with pre-mRNAsplicing regulator female-lethal(2)D [46], which was also female-biased in L. salmonis.
Overall, functional enrichment for reproduction was less clear in female-biased transcripts compared with male-biased transcripts. Many of the putative femalebiased transcripts potentially involved with reproduction had GO annotation with many other functional categories and only a small number were female-specific. For example, nuclear autoantigenic sperm protein (NASP) and histone deacetylase 1 (HD1), were female-biased in L. salmonis and have reproductive roles in similar species [54,64,65]. However, these transcripts were in the enriched GO category in utero embryonic development (p = 0.005), which is taxonomically constrained [66] and inconsistent with the virgin status of the lice used here [34]. Furthermore, the functions of NASP and HD1 differ in other species such as C. elegans in which they are important for female development and male-specific gene repression [67]. These discrepancies make it difficult to identify putative female-biased reproduction genes in the present work. In an earlier study by Eichner and colleagues [47], L. salmonis genes involved in reproduction were overexpressed in adult females compared to preadult II females. As the present study used preadult II females, it may have missed the identification of some female reproductive genes that are induced later in development than the stages evaluated here. Future studies assessing the expression and localization of these transcripts will offer insight on their exact functions. Much work remains to identify female-biased reproductive genes in L. salmonis, including the extensive changes likely to occur in female mating-responsive genes post transfer of male spermatophores (e.g. see [68,71,72]).
In general, sex-biased genes evolve faster than unbiased genes, with those exhibiting male-bias showing the highest rates of evolution (reviewed by [4]). This trend is heavily influenced by sex-biased transcripts involved in reproduction as these typically exhibit higher than normal rates of positive selection (reviewed by [4,57]). Only male-biased transcripts showed a consistently higher number of orphans in each L. salmonis population (45.1 -51.8 %) when compared to female-biased (28.3 -37.1 %) and unbiased (32.4 -34.0 %) transcripts. This trend was largely driven by the putative SFPs in this study (i.e. male-specific transcripts), of which 65.4 % were orphans. As higher rates of nucleotide substitutions are often caused by a relaxed functional constraint or greater positive selection on certain transcripts (reviewed in [4,57]), higher-resolution analyses including non-synonymous/synonymous mutation ratios (dN/dS; [70,71] and codon-usage bias [72,73] will be important in clarifying the effects of selection on these L. salmonis genes. These analyses will yield important information regarding evolutionary processes affecting reproduction, population dynamics, and drug resistance. In particular, it will be important to identify whether genes involved in drug resistance are similar to those involved in sex-biased expression as this will provide insight on molecular mechanisms behind the higher rate of drug resistance in male L. salmonis. Specifically this will inform on whether the increased resistance is due to inherent physiological factors that differ between the sexes, or to the evolutionary rate of resistance mechanisms. Sex-biased transcription related to sexually dimorphic phenotypes of L. salmonis L. salmonis exhibits extensive sexual dimorphism in morphology at the pre-adult and adult stages [27]. Several sex-biased transcripts discovered here have related functions including the male-biased transcripts enriching the GO categories cellular components involved in morphogenesis (4 transcripts, p = 0.03) and Z-disc (sarcomere; 5 transcripts, p < 0.0001). Additionally, serine proteinase stubble catalytic chain was female-specific in L. salmonis and is required for proper formation of appendages in Drosophila spp. [74]. Therefore, these transcripts represent targets for understanding sexually dimorphic morphology including features that are important in mating (described in [27,31,34]). The sexbiased orphans described here are also ideal candidates for understanding mate guarding, a common mating behavior in crustaceans, which is known to be sexually antagonistic [75]. Therefore, transcripts involved in mate guarding are unlikely to be annotated due to rapid sexual selection [75] and taxonomic constraint [50].
Other molecular links to sexual dimorphism in L. salmonis were observed in transcripts such as longitudinal lacking protein, trypsin-1, and digestive organ expansion factor, which were female-biased in this study and have roles in salivary gland, trachea, and digestive organ development and function [76,77]. Based on female L. salmonis having a greater requirement for blood in the meal [33], these transcripts will serve as important markers for understanding sexually dimorphic feeding patterns and related host-parasite interactions.
Male-biased L. salmonis transcripts also enriched several GO categories related to the sensory system, which is generally known to be more refined in male copepods [23]. These included potassium ion binding (4 transcripts, p = 0.02), solute:cation symporter activity (5 transcripts, p = 0.01), and calcium ion binding (12 transcripts, p = 0.03), among others (Additional file 3). Males are known to transfer between salmonid hosts more frequently than females [30,31] with mate location primarily being the responsibility of the male [32]. Thus, transcripts involved in the sensory system and muscle development (e.g. Z disc; Additional file 3) represent putative targets for understanding neuromuscular differences related to increased mobility in males. Furthermore, chemical cues are essential components of host and mate location in L. salmonis and serve to optimize the probability of mating through balancing the proportion of males relative to females on each host [29]. Chemosensory signaling and behavioral responses to non-host semiochemical treatments known to interfere with host recognition are sexually dimorphic in L. salmonis [32,78]. Based on the high evolutionary rates of genes involved in chemoreception [79], sex-biased orphans should be considered candidate targets for understanding the chemical ecology of L. salmonis.
The identification of sex-biased transcripts in L. salmonis also provided preliminary evidence for sexually dimorphic characteristics previously unknown in salmon lice. For example, immune-related transcripts such as rhotekin-2 and ras-related protein Rab-32 were femalespecific in this study while other related transcripts were female-biased (Additional file 2). Gene Ontology analysis also indicated a higher energy expenditure in female L. salmonis based on the female-biased expression of 15 transcripts enriching ATPase activity coupled and 60 transcripts enriching ATP binding (Additional file 3). This is a similar finding to the GO analysis of sex-biased expression in D. pulex [11], and may serve as an explanation for the lower frequency of inter-host transfer in female L. salmonis [30,31]. Although male-biased transcripts such as glycogen phosphorylase, fructosebisphosphate aldolase, and hexokinase-2 enriched the GO category glucose metabolic process (7 transcripts; p =0.02), these are likely acting as an energy source for spermatogenesis [80]. These data indicate an increased basal energy demand in female L. salmonis that is important for understanding observed sex differences in drug tolerance and resistance [35,37]. Collectively, the candidate sex-biased transcripts described here represent putative markers controlling energy expenditure, morphology, and the immune and sensory systems of L. salmonis.

Strengths and limitations of using a consensus-based approach to identify sex-bias
Recently, seven aquaporin paralogs were characterized in L. salmonis, each with different expression profiles across stage and sex [81]. Two of these aquaporins (LsGlp1_v1 (KR005660.1) and Lsaqp12L2 (KR005666.1)) showed male-specific and male-biased expression, respectively. In the present work, two male-biased contigs annotated as aquaporin 3 (BT121448.1) and aquaporin-12A (BT121051.1; see Table 3) showed more than 99 % sequence similarity to LsGlp1_v1 and Lsaqp12L2, respectively (Additional file 4). Therefore, aquaporin 3 and LsGlp1_v1 appear to be the same transcript based on sequence alignment, as are aquaporin-12A and Lsaqp12L2. Here, aquaporin 3 was only expressed above background fluorescence in one of the 58 females assayed, supporting the expression profile previously reported [81]. Additionally, aquaporin-3 was shown to be highly male-biased in the closely related C. rogercresseyi [54], suggesting this gene is important for a male-specific function in salmon lice. This type of transcriptomic consensus will be important for functional categorizations in future sea lice studies.
Within the 1407 transcripts shown to be sex-biased in Atlantic and Pacific populations, only 829 showed concordant expression profiles. The majority of transcripts in the discordant list (Additional file 2) were involved in cuticle formation and molting and, therefore, transcripts that potentially oscillate in expression levels at different molt intervals [47,82] were eliminated from this interpretation. However, the consensus identification of sex-biased transcripts in L. salmonis did prove to be over-conservative in some cases, causing particular transcripts with putative sex-bias to be overlooked.
For instance, trypsin-4 (probe: C054R168), annotated from Anopheles gambiae and involved in host seeking behavior and blood feeding [83], was female-specific in BMA2a and in Pacific L. salmonis. However, this probe did not pass quality filters in the BMA2b population and was eliminated from the consensus list. Female L. salmonis are known to feed more heavily on blood than males [33], with certain trypsins known to be involved in digestion and immune evasion on salmonid hosts [84][85][86]. Additionally, a similar transcript annotated to trypsin-1 was female-biased in this study (Additional file 2). Therefore, monitoring individual population analyses from this work is also important for identifying potential sex-biased markers in L. salmonis. Nonetheless, the consensus set of sex-biased transcripts identified here supports the characterization of L. salmonis transcript as markers for reproduction, morphogenesis, behavior, and other sexually dimorphic traits for targeted approaches (i.e. knock-out/knock-down, recombinant production, in vitro characterization, etc.) in future studies. This improved understanding of sexbiased gene expression in L. salmonis will inform future studies examining host-parasite interactions, drug resistance, reproduction, and novel drug discovery.

Conclusions
A consensus-based, meta-analysis approach was used to analyze the L. salmonis transcriptome, clearly identifying sex-biased transcripts associated with sexually dimorphic traits. Specifically, male-biased transcripts showed higher degrees of sex-bias and lower sequence similarity compared with female-biased transcripts. The enrichment of male-biased transcripts associated with reproduction was likely responsible for these trends. Our results provided insights into known and novel forms of sexual dimorphism in L. salmonis including immunity, energy expenditure, morphology, feeding, and mobility. These sexual dimorphisms will be important to consider for industry-relevant applications in areas such as parasiticidal drug response, reproductive roles, and host-parasite relationships. The current work shows that sex-biased gene expression is abundant in the pre-adult L. salmonis transcriptome and is likely to control several aspects of sexual dimorphism in this species.

Lepeophtheirus salmonis populations and collections
Adult L. salmonis were collected from Atlantic salmon aquaculture farms on the Atlantic and Pacific coasts of Canada. Two populations of Atlantic L. salmonis were collected in the spring of 2013 from Bay Management Area 2a (BMA-2a; Back Bay) and 2b (BMA-2b; Grand Manan) in the Bay of Fundy, New Brunswick (NB), as described in full previously [35,37]. A third population representing Pacific L. salmonis was collected from the Broughton Archipelago, British Columbia (BC) in 2010. Atlantic and Pacific L. salmonis are considered allopatric subspecies [38]. For all collections, egg strings were removed from adult females and larvae reared to the infective copepodid stage in static seawater hatch systems as previously described [35]. Copepodids (F1 generation) were then used to infect Atlantic salmon (Salmo salar) and allowed to develop to the pre-adult II stage. Preadult (F1) lice from all populations were used in 24 h in vitro EMB bioassays (described below) before collection and storage at -80°C for RNA extraction.

Atlantic and Pacific L. salmonis Microarray Datasets
Two microarray datasets were used to compare sex differences in L. salmonis from the Atlantic (2013 collection) and Pacific (2010 collection) coasts of Canada. The Atlantic dataset was accessed from NCBI through Gene Expression Omnibus (GEO) accession GSE56024 [37]. In this study a total of 77 pre-adult Atlantic L. salmonis, 38 females and 39 males from BMA-2a and BMA-2b were exposed to four concentrations of EMB (0.1, 25, 300, and 1000 ppb) and a seawater control, as previously reported. The bioassay protocol was identical for both populations. This study compared the effects of EMB on L. salmonis including the interactions between population (BMA-2a is more EMB-resistant than BMA-2b; [37,87]) and sex. However, baseline differences between males and females were not reported. The Pacific dataset was provided by the same laboratory group (B. Koop and S. Jones, unpublished data), which exposed 39 pre-adult Pacific L. salmonis, 21 females and 19 males, to low doses of EMB (0.01, and 0.1 ppb) or a seawater control. Lower doses of EMB were selected based on the high EMBsensitivity of this population [88]. For all F1 generation cultures, lice were maintained in filtered sea water at 10 ± 2°C and 32 ± 2 ppt. The Pacific dataset has been uploaded to GEO under the accession GSE73734.
A 38 K oligonucleotide microarray (eArray, Agilent) designed with expressed sequence tags (ESTs) from Atlantic and Pacific L. salmonis [39] was used to analyze all lice in this study. Annotation of each contig was completed using BLASTx and RPS-BLAST against SwissProt and Conserved Domain Database (e < 10 -10 ), respectively. A total of 18 -21 hybridizations for each sex and population combination (117 total hybridizations) were completed. Hybridizations were completed using methods for sample preparation, microarray hybridization, and scanning as previously reported [37,89]. Briefly, all slides were scanned using a Perkin Elmer ScanArray® at 5 μm resolution and optimized PMT intensities (1-2 % of array spots saturated). Filtering and quantification was completed using Imagene 8.1 (Biodiscovery) before completing statistical analyses in GeneSpring GX v12.6 (Agilent). A quality control (QC) filtered probe list was created for each population (Table 1) with probes included for statistical analysis only if at least 65 % of the samples in any one condition had raw fluorescent intensities ≥ 500 and showed no poor quality spots.

Sex-biased Gene Expression in L. salmonis
Microarray data was used to characterize baseline expression differences between male and female L. salmonis. Sex-biased probes were identified using a two-way ANOVA with sex and EMB as explanatory variables (Benjamini-Hochberg multiple test correction; p < 0.01; fold change (FC) ≥ 1.5). The effects of EMB on Atlantic L. salmonis transcriptomes was minor [37] and a significant transcriptomic effect was not detected in Pacific L. salmonis used here (i.e. no probes showed differential expression by EMB). The effects of EMB exposures were also controlled for in the statistical model by only using transcripts affected by a main effect of sex and through consensus-based analyses (described below). All probes with a main effect of sex for each individual population are compiled into Additional file 1.
Sex-biased probes from each population were used to create a consensus list for functional analyses. This list was limited to probes exhibiting significant and concordant sex-bias in all three populations of L. salmonis described here (Fig. 2). Sex-biased probes from individual population analysis can be found in Additional file 1, while sex-biased probes identified using the consensus sex-biased method can be found in Additional file 2. As duplicate probes represent unique contigs (i.e. transcripts) on the array [39], contig IDs are also included in the additional files. The variation between duplicate probes of consensus sex-biased transcripts is quantified in Additional file 2. Only unique transcripts are used to calculate the proportion of sex-biased expression in Atlantic and Pacific L. salmonis.
Differences in the degree of sex-bias were assessed by binning transcripts based on their degree of differential expression between male and female L. salmonis [6]. Transcripts with low sex-bias were those overexpressed by a fold change (FC) of ≥ 1.5 but < 4, while highly sexbiased transcripts had a FC ≥ 4. A mean FC value representing all three populations was also included (Additional file 2). Any transcript that did not pass the background QC filter in 100 % of the individuals within one sex was considered to be sex-specific in this study. However, based on lower limits of detection for microarrays, these transcripts may not be biologically sex-specific. A transcript similarity assessment was also completed using all QCfiltered probes against kunitz/BPTI-like toxin (probe ID: C259R052). Transcripts with similar expression patterns to kunitz/BPTI-like toxin were determined using a