Atlantic salmon (Salmo salar) aquaculture has witnessed remarkable growth over the past few decades, becoming a critical contributor to global seafood production (FAO, 2024). This growth, however, has been accompanied by persistent challenges that threaten the sustainability and economic viability of the industry. The ectoparasitic Lepeophtheirus salmonis, commonly known as the salmon louse, is currently the most significant threat to the health and productivity of Atlantic salmon farming (Whyte et al., 2014; Ugelvik and Dalvin, 2022). The economic impact of sea lice infestations on the global salmon farming industry is estimated to exceed $1 billion annually, encompassing reduced growth performance, expenses associated with treatment and prevention strategies, and direct losses through morbidity (Barker, et al., 2019). Sea lice infestation has also raised significant concerns regarding welfare of infested fish on the farm due to practices associated with delousing and potential ecological impacts on wild salmon populations adjacent to farming operations (Larsen and Vormedal, 2021).

The life cycle of L. salmonis comprises free-swimming planktonic stages (nauplius I, nauplius II) followed by the transitionary planktonic and first parasitic stage, the copepodid. Additional parasitic stages exist (chalimus I, chalimus II, pre-adult I, pre-adult II and adult) in which the parasite attaches to the host fish and feeds on the mucus, skin cells, and blood (Hamre et al., 2013). The chalimus stages are distinguished by a frontal filament that enables their secure attachment to the host, whereas the mobile pre-adult and adult stages are capable of moving across the fish’s surface, inflicting more substantial tissue damage through their aggregatory feeding activities (Fast and Braden, 2022). These various developmental stages exhibit stage-specific patterns of host-parasite interaction, with varying impacts on the host’s physiological and immunological responses (Dalvin et al., 2020; Øvergård et al., 2023). The progressive damage caused during sea lice development ultimately results in skin lesions, secondary infections, osmoregulatory disruption, and although rarer, mortality in heavily infested fish (Rodger et al., 2022).

Previous studies have revealed complex gene expression patterns that evolve throughout the course of infestation with sea lice and vary according to the parasite developmental stage (Sutherland et al., 2014; Tadiso et al., 2011). As the primary interface between the host and parasite, the salmon skin exhibits pronounced transcriptomic alterations following sea lice attachment (Krasnov et al., 2012; Sutherland et al., 2014; Holm et al., 2015; Robledo et al., 2018; Umasuthan et al., 2020; Caballero-Solares et al., 2022; Cai et al., 2022; Cai et al., 2024; Ugelvik and Dalvin, 2022; Øvergård et al., 2023). Local responses at mucosal surfaces, particularly the skin, involve upregulation of innate immune genes such as interleukins (e.g., il1b, il8), acute phase proteins, and antimicrobial peptides, often accompanied by inflammation and epithelial disruption (Skugor et al., 2008; Braden et al., 2015). These responses are typically more pronounced during the early chalimus stage, when the lice are attached to the epithelium, but they may be suppressed or dysregulated as the lice mature. In addition to localized responses at attachment sites, sea lice infestation induces systemic alterations, including modulation of immune markers, stress-related genes (e.g., heat shock proteins), and metabolic pathways, in internal tissues such as the head kidney, which functions as the primary hematopoietic organ in teleost fish (Skugor et al., 2008; Tadiso et al., 2011; Robledo et al., 2019; Øvergård et al., 2023; Valenzuela-Muñozet al., 2023; Zhong et al., 2023). Comparative transcriptomic studies have identified remarkable variation in response patterns between resistant and susceptible salmon species, providing insights into potential genetic determinants of resistance (Robledo et al., 2018; Robledo et al., 2019; Braden et al., 2023; Valenzuela-Muñoz et al., 2023; Cáceres et al., 2024; Salisbury et al., 2024). These findings hold significant implications for selective breeding programs focused on improving natural resistance to sea lice in farmed salmon populations.

In addition to the direct effects of sea lice on salmon, environmental stressors, particularly temperature fluctuations, can compound the impacts of sea lice on salmon health and immune function (Alfonso et al., 2021; Islam et al., 2022; Scharsack and Franke, 2022). Global warming and increased water temperatures are anticipated to reshape the dynamics of salmon aquaculture by simultaneously affecting fish physiology and sea lice biology (Hamre et al., 2019; Beemelmanns et al., 2021). Earlier research has indicated that elevated temperatures may exacerbate the severity of infectious diseases in Atlantic salmon, potentially altering salmon fitness, immune function and metabolic responses (Shi et al., 2019; Medcalf et al., 2021; Franke et al., 2024). In the context of L. salmonis, higher temperature has been shown to influence the parasite’s life cycle, development, and settlement success while modifying host-parasite interactions and efficacy of louse reduction, leading to enhanced infestation rates and more detrimental impacts on salmon health (Overton et al., 2019; Godwin et al., 2020; Ugelvik et al., 2022; Nilsson et al., 2023; Oldham et al., 2023). Furthermore, high temperatures can alter the stress signaling pathways and the expression of immune-related genes, potentially compromising the salmon’s immunological defenses against sea lice (Jensen et al., 2015; Nuez-Ortín et al., 2018).

The effects of infestation with different developmental stages of sea lice have been previously investigated on salmon individuals (infested versus healthy) using gene expression analyses (Skugor et al., 2008; Tadiso et al., 2011; Sutherland et al., 2014; Ugelvik and Dalvin, 2022; Øvergård et al., 2023). Prior studies suggest that systemic responses are often delayed or subdued, potentially reflecting immune evasion strategies employed by the parasite (Skugor et al., 2008; Tadiso et al., 2011; Ugelvik and Dalvin, 2022; Øvergård et al., 2023). These stage-specific and compartmentalized host responses underscore the complexity of the salmon-lice interaction and highlight the need for integrated analyses to identify meaningful biomarkers associated with the parasite’s developmental stages. However, transcriptomic differences between fish parasitized with chalimus and adult stages of L. salmonis have not been directly compared, particularly across multiple salmon families to identify consistently expressed genes associated with sea lice infestation. The present study therefore aims to remove inherent variation in this system by profiling the transcriptomic responses of the skin and head kidney of lice-infested Atlantic salmon at the family level under normal and elevated temperature conditions. The findings of this study are expected to significantly contribute to our understanding of the molecular mechanisms and biological pathways underlying the salmon’s response to sea lice infestation.

This is the first comprehensive family-based transcriptome study in Atlantic salmon in response to infestation with different developmental stages of L. salmonis. Beyond its relevance to salmon aquaculture, this study provides broader insights into the molecular mechanisms underlying host-parasite interactions in lower vertebrates. The identification of host responses to distinct developmental stages of sea lice enhances our understanding of how parasites modulate host immunity at different phases of infestation. Moreover, these findings may inform the development of targeted strategies for breeding parasite-resistant fish, improving fish welfare, and enhancing the sustainability of aquaculture systems globally. Based on within-family pairwise comparisons (T10A vs. T10C, T20A vs. T20C), a large number of genes (e.g., muc2, muc5ac, litaf, krt13, ccl20, lcn, cd22, map4k4, pbx1, trim16) were represented by paralogs showing either similar or contrasting transcriptional patterns. This observation aligns with previous findings of transcriptional divergence among duplicated genes in Atlantic salmon and may reflect functional specialization following whole-genome duplication events (Macqueen and Johnston, 2014; Lien et al., 2016; Umasuthan et al., 2020; Ignatz et al., 2024). The GO and KEGG enrichment analyses revealed terms/pathways represented by DEGs (nearly all upregulated) encoding a diverse range of enzymes/biomolecules involved in metabolism of various organic substances in the skin of fish infested with adult versus chalimus stages. These metabolic shifts facilitate the mobilization of energy substrates/stores to cope with the stress caused during infestation. In addition, the enhanced expression of several genes directly or indirectly associated with organismal stress response (ercc6, xrcc5, xrcc6, brca1, lepb2, ddb1, gadd45a, hspd1, hspa14, hsp90b1, cct8, cct6a, hif1ab, cirbp, hspa8 [hsc70], ube2v1, ube2n, sumo2) suggested a response to the more severe skin damage induced by adult forms compared to chalimus stages. These changes are consistent with elevated plasma cortisol levels and increased epithelial cell apoptosis and necrosis observed following infestation with advanced stages of sea lice (Johnson and Fast, 2004; Nolan et al., 1999).

The transcriptomic changes associated with infestation with adult versus chalimus stages of lice were remarkable in the skin. This variation was not visible in the head kidney samples from the same salmon between the two different stages. In the head kidney, no shared DEGs were identified between all families at either 10 or 20°C, and GO analyses revealed a limited number of GO terms. Based on within-family pairwise comparisons, the most frequent DEGs in the head kidney of fish infested with adult versus chalimus at both temperatures included fcrl5, cat, hbb, hbb1 and hba4 (downregulated). On the other hand, a total of 384 annotated genes (286 upregulated and 97 downregulated) were identified to be differentially expressed in the skin of Atlantic salmon infested with adult versus chalimus stages of sea lice at both 10°C and 20°C. Based on within-family comparisons, more abundant genes (present at least in 5 out of 8 within-family comparisons at either temperature, unless otherwise specified) with significant functions that were expressed in the skin of most or all families at one or both temperatures are discussed in this study, considering their expression trends/levels (see Supplementary File S6). It should be noted that one family (F265 at 10°C and F292 at 20°C) differed between temperature conditions. However, as each comparison was conducted within the same family (adult vs. chalimus stages), and the primary aim was to identify genes consistently expressed across families within each temperature, the impact of this substitution on the overall findings is likely minimal. Nonetheless, the potential influence of family-specific responses cannot be fully excluded and should be considered in the interpretation of shared gene expression patterns.

The salmon louse is known to dampen various immune-related responses in susceptible salmonids such as Atlantic salmon (Fast et al., 2006; Wagner et al., 2008; Umasuthan et al., 2020; Øvergård et al., 2023; Øvergård et al., 2025). In this study, immune responses were substantially suppressed/modulated in adult- versus chalimus-infested families. For example, the altered transcription of certain suppressor of cytokine signaling (SOCS) proteins (up: socs1; down: socs2; up-down paralogs: socs3) implied that the salmon louse could manipulate cytokine receptor signaling by recruiting SOCS proteins to circumvent the host’s immune defenses (Salisbury et al., 2024). In addition, the transcription levels of multiple genes belonging to the semaphorin family showed significant changes in the skin of families infested by adult versus chalimus stages, most of which showed lowered expressions at both temperatures (e.g., sema3ab, sema3c, sema3fb, sema4d, sema6d). On the other hand, sema3f and sema7a tended to have different paralogs with reverse expression patterns. The expression levels of plexins (plxna2, plxnb2 [LOC106584474], plxdc1 and plxdc2) and neuropilins (nrp1a and nrp2), which are known as transmembrane proteins serving as cell surface receptors for semaphorins, were also reduced at both temperatures. The enrichment of GO terms predominantly consisted of semaphorin family members (chemorepellent activity [GO:0045499] and negative chemotaxis [GO:0050919]) that are involved in stimulation of antigen-presenting cells and/or neuroinflammation (Lee et al., 2019; Rajabinejad et al., 2020; Thomas and Yang, 2023) highlighted the immunosuppression and immunoinflammatory disturbance induced by adult forms. Further immunosuppression/modulation resulting from infestation with adult compared to chalimus stages of L. salmonis at the attachment sites was supported by differential expression of several immune-related components including representatives of genes related to Toll/NOD-like receptors (up: sigirr, nlrc3l; down: tlr21, tlr5, nlrp1, nlrp12, card8, nlrc3, bcl2), nuclear factors (down: nfatc2, ikbip, nfatc1 [in 3 families]), CD molecules (up: cd276, cd44b, cd9; down: cd2, cd6, cd3e, cd8a, cd28, cd40, cd44, cd59, cd63, cd81, cd82, cd93; up-down paralogs: cd22, cd55), scavenger receptors (down: scara3, scara5, cd163) and protein kinases/phosphatases (up: tab3, mapk13, dusp4, dusp5, ptpn2b; down: map3k11, map3k20, map3k3; up-down paralogs: map3k5).

The proteinaceous (i.e., trypsin and cathepsin L) and non-proteinaceous (i.e., prostaglandin E2, PGE2) products secreted by the salmon louse are known to interfere with the host’s immune responses and facilitate the flow of blood to the attachment sites (Johnson et al., 2002; Fast et al., 2004; Fast et al., 2007; Firth et al., 2000; McCarthy et al., 2012). Based on within-family comparisons, three subtypes of prostaglandin E2 (PGE2) receptors (EP1, EP2 and EP4 encoded by the genes ptger1 [down], ptger2 [up-down paralogs] and ptger4 [down]) along with prostaglandin D2 receptor 2 (ptgdr2, up) and prostaglandin F2 receptor negative regulator (ptgfrn, up) were impacted in families infested with adult compared to chalimus stages of lice. Upregulation of the subtype EP2 of PGE2 receptor has been previously reported in the skin of Atlantic salmon infested with copepodids of lice compared to non-infested fish (Øvergård et al., 2023). The reduced expression of EP receptors in fish infested by adult lice may be a consequence of chronic exposure to secretary products (i.e., PGE2) of the salmon louse, resulting in their desensitization or internalization (Malty et al., 2016). The enhanced expression of pla2g4c, ptgis, ptgs2b, lta4h, and ltb4r, which are involved in lipid mediator synthesis, vasodilation, and/or leukotriene signaling, was observed in nearly all families, indicating a heightened inflammatory response at sites infested by adult lice compared to chalimus stages (Murakami et al., 2003; Ricciotti and FitzGerald, 2011). Dampened expression of apc, encoding angiotensin-converting enzyme that promotes vasoconstriction, in all families seemed to be a feedback mechanism to control excessive inflammation. This, however, could allow the parasite to cause chronic infestation by impairing the host’s immune ability. In addition, several other genes correlated with inflammation and inflammasome signaling were also differentially expressed in the skin of most families (up: gpr84; down: nlrp1, nlrp12, card8, nlrc3, up-down: nlrp3, usp7 and usp47). The increased expression of gpr84 that promotes the release of inflammatory cytokines and reduced expression of nlrp12, card8 and nlrc3 that negatively regulates inflammation was indicative of excessive inflammatory responses (Zheng et al., 2020; Zhang et al., 2022). On the other hand, a decreased level of nlrp1, which is involved in normal release of inflammatory cytokines, accompanied with fluctuated expression of nlrp3 and its regulators (usp7 and usp47) could lead to a dysregulated status contributing to chronic inflammation or suppressed immune responses (Palazón-Riquelme et al., 2018).

The expression of several mediators associated with acute inflammation were found to be reduced in the skin of fish parasitized with adult compared to chalimus stages of lice (crp, hp, a2m, ltc4s). Conversely, saa5 showed higher expression in the skin of most families at both temperatures. Earlier findings highlighted the upregulation of saa5 in Atlantic salmon infested by various stages of lice (Sutherland et al., 2014; Braden et al., 2015; Ugelvik and Dalvin, 2022), suggesting that persistent sea lice infestation could result in chronic inflammation, characterized by prolonged expression of pro-inflammatory cytokines such as saa5. In addition, higher transcript levels of il1b (LOC100136449 and LOC106570815), il4i1, il1r2 and sigirr (il-1r8) were noticed in most/all families at both temperatures, while il4/13a and il17d showed reduced transcript expression. Increased mRNA levels of il1b and il8 have been previously shown using qPCR analysis in the skin of both Atlantic salmon and rainbow trout following infestation with juvenile to advanced stages of sea lice including those with no blood-feeding activity (copepodid and/or chalimus) (Dalvin et al., 2020; Ugelvik and Dalvin, 2022). The upregulation of il1b (pre-adult-infested vs. non-infested) and reduced expression of il4/13a (adult vs. chalimus) was also reported in the skin of Atlantic salmon in a previous study (Øvergård et al., 2023). This unregulated expression of il1b could lead to excessive or prolonged inflammation, as augmented transcriptional levels of il1r2 (a decoy receptor that antagonizes il1b) and sigirr (a negative regulator of TLR/IL-1R signalling) were detected in almost all families, possibly to dampen il-1β-mediated signaling and protect skin tissue from potential damage (Qin et al., 2005; Schlüter et al., 2018). Interleukin-4 induced 1 (il4i1) is an L-amino acid oxidase that produces immunosuppressive metabolites by catabolizing essential amino acids required for T-cell activation/proliferation, and thus, its higher transcript level could be regarded as immunosuppression caused by advanced stages of sea lice (Hirose et al., 2024). The lowered expression of il4/13a and il17d, which are involved in protection against parasites, was indicative of compromised immunity, tissue healing and barrier function (Sequeida et al., 2020; Huangfu et al., 2023). The expression profile of il8 was found to be different among families (down in 3 families at 10 and up in 1 family at 20). This could be due to the varied expression level of il8 in fish infested by adult or chalimus stages, since this gene was shown to be expressed (upregulated) in both fish infested by chalimus and adult stages of lice (compared to un-infested fish) in a previous study (Ugelvik and Dalvin, 2022).

The transcription of several chemokines and their ligands/receptors (up: ackr4b, ccr3, cxcr1 and cxcr2; down: ackr3, ackr3b, ackr4, ccr7, ccl8, ccl13, ccl17, ccl21, ccl25 [only at 20], ccl25a, ccl27a and cxcl12a; up-down paralogs: ccl4 and ccl20) significantly differed between families infested by adult compared to chalimus stages. Similar chemokine-related transcriptomic changes have been previously documented for fish infested by advanced (pre-adult female and adult male) forms of sea lice (Cai et al., 2024). Contrary to inconsistent expression of il8, the transcription of il8 receptors (cxcr1 and cxcr2) that are primary stimulants of neutrophils was enhanced (3-6-fold changes) in most families, potentially contributing to uncontrolled neutrophil responsiveness. Two gene copies related to atypical chemokine receptor 4 represented opposite transcription patterns (up: ackr4b; down: ackr4 [LOC106568919]). Atypical chemokine receptor 4 (ackr4), which is expressed primarily in non-immune cells such as keratinocytes, is known to bind and internalize the chemokines ccl19, ccl20, ccl21 and ccl25, thus regulating the migration and positioning of immune cells (dendritic and T cells) expressing the chemokine receptors ccr6 (ligand: ccl20), ccr7 (ligands: ccl19 and ccl21) and ccr9 (ligand: ccl25) (Meyrath et al., 2021). Likewise, ackr3 internalizes and serves as a scavenger receptor for cxcl11 (receptor: cxcr3) and cxcl12 (receptors: cxcr4 and cxcr7), modulating their availability (Cuesta-Margolles et al., 2024). In addition, the ccr3 gene encodes a protein primarily found on eosinophils and a subset of Th₂ lymphocytes, serving as a receptor for chemokines such as eotaxins (ccl11 and ccl26), monocyte chemoattractant protein-3/4 (ccl7, ccl13) and RANTES (ccl5) (Manousou et al., 2010). The dysregulated/contrasting expression of several members of the chemokine family, known for their involvement in directing immune cell chemotaxis, in the skin of fish infested by adult versus chalimus stages of lice could favor the recruitment of specific immune cells that aid sea lice persistence. The immunomodulatory effects of salivary products of blood-feeding ticks on chemokine activity of immune cells have been previously reported in terrestrial-associated models in vivo and in vitro (Vančová et al., 2007; Wang et al., 2007; Oliveira et al., 2008; Langhansova et al., 2015). The association of ackr4 and ccr9 in resistance against the ectoparasite Ichthyophthirius multifiliis has been also suggested in Colossoma macropomum using a genome-wide association study (Lira et al., 2023). These results highlight the importance of identifying louse-derived secretary products that influence the chemokine signalling system in Atlantic salmon to develop possible parasite-protective vaccines.

Some members of the tumor necrosis factor (TNF) cytokine family represented modulated expressions in almost all families (up: tnfrsf6b and tnfrsf9 [LOC106572390]; down: cd40, fas, tnfsf11 [LOC106582173], tnfsf12, tnfrsf14 and traf3ip3). Paralogs with opposite transcription patterns were identified in all families for litaf, a DNA-binding protein that contributes to the progression of inflammation by altering cytokine levels and plays a role in the p53-induced apoptotic pathway (Zou et al., 2015). The transcription of several DEGs associated with apoptosis (up: aifm1, bax, casp3b, casp3, perp, malt1, tp53, api5, tigara; down: bcl2, bcl11ba, bcl6b, prf1, gzma, phlda1, camk4), coupled with enrichment of the GO term “p53 signaling pathway”, indicated the induced/dysregulated apoptotic response in damaged/infested cells in families infested (adult vs. chalimus) (Wang H. et al., 2023). The transcript levels of genes associated with interferons showed varied expressions in adult- vs. chalimus-infested families (up: ifnar1a, ifngr1a, ifngr1b, irf6, irf7, irf8, ifi44, ifit1, ifrd2, sting1; down: ifi30; up-down: ifit5). Genes related to DEAD-box helicases (up: ddx3x, ddx5, ddx19a, ddx55, ddx56), which act as sensors for dsRNA, cytosolic DNA and viral RNAs (Taschuk and Cherry, 2020), exhibited higher expression in families infested by adult forms. In addition, some TRIM proteins involved in host antiviral defense and/or regulation of interferon signaling (up: ftr82, ftr83, trim14, trim39, isg15, uba7; down: trim9, trim21, trim25, trim35) showed altered expressions (van Gent et al., 2018; Wang and Ning, 2021). Modulation of viral-responsive genes has been previously reported in fish infested by various developmental stages of lice (Umasuthan et al., 2020; Cai et al., 2022; Caballero-Solares et al., 2022; Øvergård et al., 2023). Motile-stage sea lice are known to carry divergent RNA viruses, which might account for modulation of interferon-related markers in the skin of Atlantic salmon (Økland et al., 2014; Chang et al., 2023). In addition, two paralogs related to cyclic GMP-AMP synthase gene (cgas and cgasa) showed higher expression in almost all families. cgas is a cytosolic DNA sensor that detects aberrant/foreign DNA from invading pathogens, resulting in type-I interferon production via the cGAS-STING pathway (Ou et al., 2021).

Two paralogs (LOC100136453 and LOC100136439) of the cathelicidin antimicrobial peptide (camp or ll37) gene were identified to have higher expression in the skin of fish infested with adult compared to chalimus stages, one of which was shared among all families at both temperatures (LOC100136453). In contrast, defensin, beta-like 1 (defb1) gene showed lower expression in most families. Similar changes in expression have been also observed in Atlantic salmon following sea lice infestation (Krasnov et al., 2015). Increased expression of camp (asCATH2) in lice-infested fish was suggested to be a host signal that facilitates the recognition of host by the parasite (Núñez-Acuña et al., 2018). The association of elevated level of camp with modulated expression of B-cell lymphoma 3 (bcl3), nuclear factor kappa B (NF-κB) and vitamin D receptor (VDR) has been already demonstrated in human keratinocytes (Büchau et al., 2009; Park et al., 2011). A variety of secreted gel-forming and transmembrane mucins (up: muc17; down: muc19; up-down: muc2, muc3a and muc5ac) were differentially expressed in most/all families. In addition, inconsistent transcript levels of fcgbp encoding a mucin-like protein that interacts with muc2 to preserve the integrity of the mucosal layer were detected among families (Liu et al., 2022). The modulation of muc2, muc4 and muc5 has been previously reported in lice-infested Atlantic salmon (Caballero-Solares et al., 2022; Gao et al., 2024; Salisbury et al., 2024). The mucus layer contains protective and signal-delivering mucin glycoproteins that are typically secreted upon exposure to various stressors and pathogens (Kufe, 2009). The glycan-rich molecules present on the surface of lice or in their secretions may interact with host mucins by modifying and/or mimicking their structures to persist on the host. Representatives of lectin-associated genes differentially expressed in the skin of families infested with adult versus chalimus stages of lice included: asal (up); mrc2, pla2r1, colec12, clec10a [mgl], clec3ba, clec4e [mincle], clec4m [cd209l], cd209a, cd209c, cd209e, zg16, lgals4, lgalsl and lgalsla (down); cd22, mrc1 and siglec1 (up-down). Despite enhanced expression of the asal gene encoding a poorly-characterized mannose-specific lectin, the majority of C-type (CLECs), S-type (galectins) and I-type (siglecs) lectins that are involved in recognition/presentation of pathogen-specific biomolecules such as carbohydrate and chitin represented reduced expression in most families, suggesting persistent immune evasion by the parasite during infestation (Varki and Angata, 2006; Cote et al., 2017; Sousa et al., 2024). In lice-resistant salmonid species (i.e., Coho salmon), multiple classes of C-type lectin receptors (clec6a, clec4e, and cd209) are strongly overexpressed in early stages of infestation, which facilitates the rejection of the attached parasite (Braden et al., 2023). The expression of nccrp1, serving as a receptor-like protein expressed in non-specific cytotoxic cells (NCCs), was enhanced in almost all families. nccrp1 is believed to play a role in the recognition of cells infected by pathogens and activation of cytotoxic responses by activating intracellular signaling pathways, notably the JAK-STAT pathway (Sukeda et al., 2023). This gene has been reported to bind to the receptors of some protozoan parasites and bacterial pathogens (Sukeda et al., 2023; Teng et al., 2023). Several genes related to the complement system were modulated in adult-compared to chalimus-infested families (up: c1qbp, cfh and c5ar1; down: c1r, cd93, c4, c4b and cfd; up-down: c3 and cd55). c1qbp encodes a multifunctional protein involved in mitochondrial fitness, inflammation, apoptosis regulation and ribosome biogenesis (Wang et al., 2022). The elevated expression of c1qbp may represent a regulatory mechanism for cGAS activation, as its higher expression was observed in all families (Song et al., 2021). Complement factor H (cfh) is an essential inhibitory regulator of the alternative pathway activation, whereas c5ar1 stimulates chemotaxis in response to C5a (Coulthard et al., 2018; Saxena et al., 2024). All complement-relevant genes with lowered expression were found to be involved in activation of the complement system and innate immune responses. Taken together, these changes implicated the possible suppression/dysfunction of components correlated with complement pathways.

Genes encoding major histocompatibility (MHC) antigens/molecules (down: bl3-7, h2-q10, mr1, rt1-b; up-down: h2aa) showed differential expression in adult- versus chalimus-infested families. MHC class I and II proteins play a pivotal role in the cell-mediated adaptive immune responses, and their reduced/modulated expression suggest diminished immune function (Wieczorek et al., 2017). The expression of immunoglobulin-associated genes was modulated in families infested with adult versus chalimus stages (up: vsig10l and vsig8b; down: kbas, mopc-321, lrig1 and ighd; up-down: pigr). Many of the members of the V-Set and immunoglobulin (Ig) domain-containing (VSIG) protein superfamily represent immunosuppressive properties. For example, both vsig8b and vsig10 can suppress T-cell proliferation and cytokine production (Zhou et al., 2022). Depressed transcript levels of lrig1 and ighd may induce chronic inflammation and reduce antigen binding activity, respectively (Nakamura et al., 2013; Qiu et al., 2021). The gene pigr encodes a receptor protein that transports polymeric immunoglobulins (IgM) to the mucosal surface of epithelial cells, and its regulation is controlled by interferons and cytokines (Kong et al., 2018). Some genes belonging to the butyrophilin family showed lower expression in most/all families (btn1a1, btn2a2, btn3a1, btn3a2, btnl2). Butyrophilins belong to the immunoglobulin superfamily and are associated with the MHC complex, primarily serving as co-signaling molecules involved in T lymphocyte regulation (Malinowska et al., 2017). Most aforementioned butyrophilins (except btn3a1) represent inhibitory functions on T-cell proliferation and/or cytokine/interferon production, and their reduced expression may impair tissue repair and contribute to chronic inflammation (Lebrero-Fernández et al., 2016). The expression of T-cell (up: vtcn1, mal and pbk; down: gzma, cd2, cd6, cd28, nfatc2, rftn1, rftn2, trac, trbc2, trb and cabin1) and B-cell (up: bcl3 and bcl10; down: bcl6b, pbx1 and pbxip1; up-down: bcl11b and cd22) relevant transcripts was modulated in the skin of salmon infested by adult versus chalimus. As an immune checkpoint molecule, vtcn1 negatively regulates the immune reactions by inhibiting T-cell activation/proliferation and cytokine production (Vaishnav et al., 2022). Reduced expression of receptors/antigens essential for T-cell activation, proliferation and/or differentiation (gzma, cd2, cd6, cd28, trac, trbc2, trb) and nfatc2 that is a transcription factor implicated in induced expression of cytokine genes in T-cells indicated the repressed adaptive immune responses (Peng et al., 2001; Antonacci et al., 2020; Tian et al., 2022). bcl10 is a central component of the card11-bcl10-malt1 (CBM) signaling complex, which links antigen receptor signaling to NF-κB activation. Likewise, bcl3 is a context-dependent regulator of T cell responses that controls NF-κB activity (Herrington and Nibbs, 2016). bcl3 was reported to alter cytokine secretion profile by dendritic cells in response to carbohydrate-expressing pathogens like Schistosoma mansoni (through interaction of the parasite sugar ligands with cd209 [dc-sign]), leading to inhibited expression of pro-inflammatory cytokines but enhanced expression of il10 and chemokines capable of attracting Th₂ cells (Gringhuis et al., 2014). Similar anti-parasitic responses were also reported in Coho salmon, where Th₁-type response was associated with lice rejection (Braden et al., 2023). The lack of such responses in Atlantic salmon makes this species prone to infestation with sea lice (Sveen et al., 2025).

Representatives of markers associated with other immune cells (neutrophils, monocytes/macrophages, mast cells and dendritic cells) that were differentially expressed in families infested by different developmental stages of lice included: lect2, ncf2, arg1, arg2 and lifr (up); lsp1a, aif1, kit, kita, csf1a and mrc2 (down); serpinb1, mrc1 and mst1r (up-down). Of the two lect2 genes existing in teleost fish, lect2-b is highly expressed in mucosal organs, such as the skin and intestine (Hu et al., 2022). Enhanced expression of lect2, a multifunctional protein that acts as a chemotactic factor (Lao et al., 2024), was also reported in Atlantic salmon infested with early to advanced stages of sea lice (Skugor et al., 2008; Gallardi et al., 2019; Øvergård et al., 2023). Co-stimulation of arg1 and arg2 may contribute to T-cell hyporesponsiveness as a result of depletion of L-arginine required for T-cell proliferation and function (Martí i Líndez and Reith, 2021). mst1r (ron), mainly expressed in the tissue-resident macrophages, inhibits inflammation and promotes wound healing (Dai et al., 2016). The reduced expression of mst1r (loss of ron signaling) in most families could result in prolonged inflammation, impaired tissue repair and increased susceptibility to infestation (Hunt et al., 2023). Unlike mcr1 which is more specialized for pathogen recognition, mrc2 is involved in collagen turnover process (Melander et al., 2015). The reduced expression of mrc2 concurrent with modulation of its receptor, i.e., plaur (up-down at 10°C and up at 20°C in all families) suggested the potential changes in extracellular matrix (ECM) remodeling and cellular migration (Zhao et al., 2022). serpinb1 is a protease inhibitor that regulates neutrophil serine proteases, protecting tissues from excessive damage during inflammation (Choi et al., 2019). The transcript levels of several genes corresponding to melanogenesis, melanocyte regulation, melanocyte survival, melanosome transportation and/or skin pigmentation (e.g., mc5r, mlph, mreg, mitf, sox4a, sox10, pax3, pax7, pax7a, adamts20, rab27b, rab38, kitlg, kitlga, kit and kita) were found to be reduced in adult-compared to chalimus-infested fish (Kawakami and Fisher, 2017; Xu et al., 2020; Myung et al., 2021; Sevilla and Grichnik, 2024). The kit receptor tyrosine kinase and kit ligand have been duplicated in teleost, where kita and kitlga are the paralogs associated with melanogenesis (Braasch et al., 2007; Hultman et al., 2007). Melanin-based pigmentation was suggested to be correlated with immunocompetence and resistance to ectoparasites in Atlantic salmon (Kittilsen et al., 2012). kit/kita receptors are mainly expressed on melanocytes and mast cells, and their decreased expression might be associated with enhanced levels of il4 and adrenocorticotropic hormone (ACTH) that suppress melanin production (Pillaiyar et al., 2017).

The wound healing process in fish involves four overlapping phases: re-epithelialization, inflammation, granulation (repair) tissue formation and tissue remodelling (Sveen et al., 2020). Prolonged parasitic infestations can lead to a sustained inflammatory response, which hinders the process of wound healing and promotes the development of chronic wounds (Skugor et al., 2008; Gumede et al., 2024). Chronic wounds are characterized by persistent inflammation (elevated levels of neutrophils and pro-inflammatory macrophages), a reduced proliferative phase and increased proteolytic activity (Wilkinson and Hardman, 2020). The transcription level of a large number of genes involved in ECM architecture and wound healing process/regulation was modulated in adult-compared to chalimus-infested families (up: tmprss13b, anxa1, panx1, cd44b, plat, ctnnb1, errfi1, pim1, met, myl6 and tpm3; down: eln, ogn, krt18a, krt20, krt8.2.l, hpse2, prss16, cd44, ctsw, ctsl, serpine1, dact1, raf1, adra2b, ret, mras, mdkb, ccdc80l2, ppara and pparb2b, myh10; up-down: col1a1, col6a2, krt13, krt18, krt8, mylk and myo1c). The majority of structural and non-structural components of ECM revealed reduced expression. For instance, there were 13 DEGs belonging to the collagen family (col1a1a, col1a1b, col1a2, col2a1, col2a1b, col5a1, col6a3, col7a1, col10a1, col10a1a, col11a1, col16a1 and col27a1b) that consistently showed reduced expression in all families at both temperatures. In addition, matricellular proteins of different families (CCN, SPARC, ENPP, fasciclin, fibulin, olfactomedin, R-spondin, tenascin, thrombospondin) mainly had decreased transcription levels (down: ccn1, ccn2, ccn4, thbs2a, thbs2, tnn, tnxb, postna, postn, sparc, smoc2, sparcl1, rspo3, spon1, spon2, fbln7, abi3bp, enpp2 and adgrl2a; up-down: thbs1 and smoc1). Unlike structural ECM proteins, matricellular proteins primarily function as modulators of angiogenesis, inflammation and ECM assembly, with their expression significantly increasing at the site of injury to facilitate the repair process (Cárdenas-León et al., 2022). The reduced expression of both structural and non-structural ECM components in the skin can lead to compromised skin integrity, promoted chronic inflammation and delayed wound healing (Cárdenas-León et al., 2022). Transglutaminases (TGs), also known as protein-glutamine γ-glutamyltransferases, catalyze the formation of amide bonds between proteins to form insoluble crosslinked protein structures resistant to proteases degradation (Martins and Choupina, 2018). The expression of some genes related to the transglutaminase family was modulated in most/all families at both temperatures (up: tgm2, tgm3, tgm1l1 and tgm5l; up-down: tgm1). tgm1, tgm3 and tgm5 are expressed in epithelial tissue and involved in cell-envelope formation during keratinocyte differentiation, whereas tgm2 is produced in different tissues and plays a role in multiple processes such as wound healing and apoptosis. The induced expression of TGs is associated with extremely stressful conditions and mucosal inflammation/epithelial lesions (Lorand and Graham, 2003; Mehta, 2005). The enrichment of the GO term “keratinization” consisted of representatives of TGs and keratins (krt8.2.l and krt8) revealed the possible protective role of epidermal keratinization against infestation caused by adult lice. In addition, some genes encoding proteins with important roles in skin barrier function (up: degs2, cldn7b, aloxe3; down: cldn3c, cldn4, cldn5, cldn5a, pnpla1 [only at 10°C]) were modulated in most/all families. degs2 encodes a bifunctional enzyme that produces ceramides, and aloxe3 encodes the eLOX3 enzyme that is involved in the production of oxylipins (Krieg et al., 2013; Song et al., 2023). The enhanced expression of degs2 and aloxe3 could maintain lipid balance and contribute to the permeable barrier function of the skin against infestation with adult stages. The altered expression of claudins that play a crucial role in maintaining the integrity of tight junctions could result in dysregulated epithelial permeability (Angelow et al., 2008).

A group of matrix metalloproteinase enzymes (up: mmp9 and mmp13; down: mmp15, mmp16 and mmp23bb; up-down: mmp14 and mmp19) demonstrated significant transcriptional changes in the skin infested by adult versus chalimus stages. mmp9 and mmp13 respectively degrade type IV and type II collagens as their preferred substrates (Yabluchanskiy et al., 2013; Hu and Ecker, 2021). mmp9 (gelatinase B) is secreted by a variety of cell types, including neutrophils, macrophages, and skin keratinocytes/fibroblasts (He et al., 2023), and it can increase mRNA levels of pro-inflammatory cytokines and modulates chemokine signaling, resulting in impaired vascular remodelling and delayed resolution of skin inflammation (Yabluchanskiy et al., 2013). mmp13 (collagenase 3) which is produced by keratinocytes, fibroblasts and macrophages is abundantly expressed in chronic wounds but not in acute wounds and the normal skin epidermis, suggesting its role in delayed wound healing process (Vaalamo et al., 1997; Saarialho-Kere, 1998). The systematic increase of mmp9 and mmp13 has been suggested as a hallmark of transit from acute to chronic inflammation following sea lice infestation (Skugor et al., 2008; Tadiso et al., 2011). Wound-mediated induction of proteinases can not only break down several components of the ECM but also modulate the activity of growth factors (Stamenkovic, 2003). In this regard, multiple genes encoding growth factor-related peptides (TGF-β, IGFs or other growth factors) that have important roles in various phases of wound healing exhibited variable expression levels in adult- versus chalimus-infested families (e.g., tgfb1 [in 4 families], fgfbp1, hbegf, vegfaa [up]; tgfb2, tgfb3, tgfbr1, ltbp4, igf1, igfbp5, pdgfc, pdgfa [down]). In addition, some genes associated with BMPs, SMAD and WFIKKN proteins (e.g., bmp2, bmp4, bmp7, smad6, smad7, wfikkn2) represented lower expression. bmp2 and bmp4 promote angiogenesis and tissue remodeling, whereas smad6 and smad7 respectively function as inhibitory regulators of BMP and TGF-β signaling pathways to maintain tissue homeostasis (Goto et al., 2007; Dyer et al., 2014). wfikkn2 encodes a companion protein that regulates the balance and signaling activity of growth factors such as tgfb1, bmp2 and bmp4 (Szláma et al., 2010; Monestier and Blanquet, 2016). A variety of genes encoding proteins belonging to ADAMs (up: adam8, adam9 and adam28; up-down: adam10), ADAMTSs (down: adamts2, adamts2a, adamts5, adamts10, adamts17, adamts20), TIMPs (up: timp2; down: timp3) and TRIM (e.g., trim9 [down]; trim16 [up-down]) were differentially expressed in the skin of adult- versus chalimus-infested families. The increased expression of adam9 may impair keratinocytes migration/proliferation and wound re-epithelialization (Chou et al., 2020), delaying wound closure. adam10 sheds cell surface proteins such as Notch receptors and mediates E-cadherin shedding in fibroblasts and keratinocytes (Maretzky et al., 2005; Sezin et al., 2022). The elevated levels of ADAMs may contribute to delayed healing by excessive tissue breakdown, promoting sustained inflammation and dysregulated signaling (Wang Z. et al., 2023). Reduced levels of the abovementioned ADAMTSs proteins that are involved in collagen maturation (adamts2), regulation of ECM structure (adamts5) and formation of microfibrils (adamts10 and adamts17) can lead to chronic wounds, ultimately reducing tissue integrity and delaying the healing process (Kelwick et al., 2015; Karoulias et al., 2020). trim9 regulates cytoskeletal dynamics and cell migration (Menon et al., 2021), which are crucial for the reorganization and closure of wounds. trim16 has been shown to be an important regulator of keratinocyte differentiation and proinflammatory cytokine secretion (Ren et al., 2020). The misregulation of TRIM proteins can contribute to improper wound healing and chronic wound pathology.

The expression of genes related to signaling pathways involved in wound healing such as TGF-β, Notch, Wnt/β-catenin, ErbB, Ephrin/Eph, PI3K/Akt, HIF-1, JAK/STAT, MAPK and focal adhesion has been reported to be dysregulated in chronic wounds (Shi et al., 2015; Leyane et al., 2021; Basu and Martins-Green, 2022; Bonnici et al., 2023). In this study, representatives of genes directly or indirectly associated with JNK/Wnt (up: hif1ab; down: wnt3, wnt5b, wnt6, wnt7a, mapk8 [jnk1], mycb, up-down: lbh), HIF-1 (up: hif1ab, vegfaa, tgfb1 and nos2b [inosb, in 4 families]; down: usp20), Ephrin/Eph (up: ephb4a, epha2 and efna5; down: epha3, epha4, epha7, ephb2, efnb3, efna2 and efna5b; up-down: efnb1), ErbB (down: erbb3 [in 4 families]), Notch (down: dtx1), JAK/STAT (down: stat1, stat5a, stat5b), MAPK (up-down: mapkapk2 and map4k4) and focal adhesion (up: lama3, lamb3, lamc2, itga2, itgb3; down: lama4, itga5, itga1, itgal, itgb1 [cd29]) signaling pathways were identified in adult-compared to chalimus-infested families. In addition, several members of semaphorins, ephrins, netrins (ntn1, ntn1a, ntn2 and ntn3) and slit proteins (slit1, slitrk6, srgap), represented mainly by reduced transcript levels, were enriched in GO/KEGG pathways associated with axon guidance. Axon guidance molecules have been shown to regulate cell migration, angiogenesis and tissue remodeling, and their dysregulation contributes to delayed/impaired wound healing processes (Chilton, 2006; Nakanishi et al., 2022). The increased expression of hif1ab that is stabilized during hypoxia concurrent with deceased expression of hemoglobin subunits (hbb, hba4, hba1, hbb1) that are involved in oxygen transport and binding suggested the possible hypoxic environment in infested sites, which may contribute to wound persistence and delayed healing. Several genes encoding lysyl oxidase (lox) and lox-like (loxl) proteins showed decreased expression in families infested by adult compared to chalimus stages (loxa, lox, loxl2a, loxl2b, loxl3b, loxl4). Lysyl oxidase (lox) proteins facilitate the cross-linking of elastin and collagen fibers, and their altered/dysregulated expression is linked to skin pathologies (Szauter et al., 2005; Laczko and Csiszar, 2020).

As indicated by enrichment analyses, GO terms and KEGG pathways obtained based on comparisons under elevated temperature conditions encompassed a more diverse and frequent set of genes. While this study provides important insights, further research is required to determine whether these gene modulations are driven by elevated temperature, increased lice pathogenicity, or both, as this could not be fully addressed within the scope of the present work. Based on RNA-seq analysis, the transcriptomic responses of salmon families with different lice burden to infestation with adult compared to chalimus stages of lice were not highly dependent to temperature (10/20°C), as the majority of the identified genes were significantly expressed at both temperatures. In addition, the expression patterns resulted from comparisons between families infested by adult versus chalimus stages of sea lice were mostly identical at both thermal conditions. On the other hand, the expression level of genes identified based on these comparisons showed important differences. For example, among the genes expressed across all families, several (e.g., gsr, cox2, ptgis, il1b, pla2g4c, c1qbp, mmp9, mmp13, casp3b, perp, tigara, ftr83, camp, itga2, tubb2a, tubd1 and gdpd3) exhibited reduced upregulation, while others (e.g., tgfb3, adamts17, adamts20, camk1a, trim9, col16a1, col2a1b, col10a1a, fndc1, ncana and ltbp4) represented less pronounced downregulation in F361 at both temperatures. serpinb1 and hspa8 were among DEGs with paralogs exhibiting opposite expression patterns, which showed reduced up- and downregulation in F361 compared to other families. Moreover, several genes related to tissue repair (e.g., mrc2, lama3, lamc2, efna5b, serpine1, wnt5b, itga1, ret, ptk2ba, ptk6, ptprj), growth factor signalling (stat5a, stat5b, smad6, smad7, s100a13), inflammatory response (e.g., nlrp1, bmp7, usp7), melanogenesis (kita), stress/DNA damage response (e.g., mao, maoa, api5, dnaja, dnajb6b, hsp10, trim39, scara3, mgst3) and immune system (e.g., cd209e, sema3a, sema4d, sema6a, sema7a, plxdc1, btn2a2, btn3a1, nfatc2, c1r, cd40, tnfsf10, tnfsf11, tnfsf12, tnfrsf14) were not expressed in F361, while they had modulated (up or down) expression in other families. The qPCR analysis also revealed important differences in the expression level and pattern of col1a1, mmp9 and saa5 at the attachment site (skin) between F361 and other families at both temperatures. These results collectively suggest that genes associated with immune-inflammatory response, tissue repair, apoptosis, cell adhesion and heat shock response are associated with potential differences among salmon families to sea lice infestation.

This study presents a novel transcriptomic investigation of Atlantic salmon responses to sea lice infestation, utilizing RNA-seq analysis across multiple salmon families to unravel the complex molecular mechanisms underlying host-parasite interactions. By analyzing the global gene expression profiles of salmon families in response to different sea lice developmental stages (adult vs. chalimus) under both normal and elevated temperatures, we have unveiled a substantially more intricate molecular response than previously recognized. The complexity of transcriptomic responses to sea lice infestation in Atlantic salmon is further reflected by the contrasting transcriptional patterns observed among multiple gene paralogs, suggesting possible functional diversification among closely related genes. This genomic complexity further complicates the molecular response mechanisms employed by Atlantic salmon when confronting pathogens such as sea lice. Our findings reveal significant dysregulation of multiple genes and biological pathways, most notably those associated with inflammatory responses, immune system functioning, and wound healing processes. These disruptions fundamentally compromise the host’s ability to mount an effective defense, ultimately contributing to the persistence and survival of sea lice on susceptible hosts. The induced transcriptomic alterations suggest a multifaceted strategy adopted by sea lice that undermines the host’s defensive capabilities.

Interestingly, our analysis indicates that the transcriptomic response to sea lice infestation was not highly temperature-dependent, although elevated temperature potentiated metabolic and stress responses and may potentially exacerbate the impact of infestation over prolonged exposure. This study demonstrates that family-based transcriptomic analysis is a powerful tool for identifying specific molecular markers associated with sea lice infestation and host resistance, which could be used for breeding more resilient salmon populations with improved defense against sea lice infestation. Overall, this research has enhanced our understanding of the molecular intricacies underlying host-parasite interactions in Atlantic salmon, providing a foundation for future research and potential practical applications in sea lice control. The RNA-seq data generated in this study can support genomic selection for sea lice resistance in salmon breeding programs through integration with genome-wide association studies (GWAS) and the application of weighted correlation network analysis (WGCNA) to identify gene modules associated with infestation.