Local inflammation at the salmon louse (Lepeophtheirus salmonis) attachment site drives copepodid rejection in Coho Salmon (Oncorhynchus kisutch)

Local inflammation at the salmon louse (Lepeophtheirus salmonis) attachment site drives copepodid rejection in Coho Salmon (Oncorhynchus kisutch) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Local inflammation at the salmon louse (Lepeophtheirus salmonis) attachment site drives copepodid rejection in Coho Salmon (Oncorhynchus kisutch) Lene Sveen, Mark D. Fast, Torstein Tengs, Gerrit Timmerhaus, Marianne Vaadal, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4947433/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Cell and Tissue Research → Version 1 posted 13 You are reading this latest preprint version Abstract Salmon louse ( Lepeophtheirus salmonis ) poses significant risks to both wild and farmed salmon populations. Atlantic Salmon (Salmo salar) are highly susceptible to the parasite, while Pacific salmon species such as Pink ( Oncorhynchus gorbuscha ), Coho ( Oncorhynchus kisutch ), and Chum ( Oncorhynchus keta ) salmon exhibit varying levels of resistance. This study explored L. salmonis infestation dynamics in four salmonid species (Atlantic, Pink, Coho, and Chum Salmon) across three challenge trials using the same fish cohort. Infestation was only successful in the third trial, where sedation facilitated parasite attachment. Skin and fin samples were collected at 12, 24, 36, 48, 60, and 168 hours post-infestation (hpi) during the early infestation phase. Results showed low L. salmonis settlement on Chum and Pink Salmon, while Coho Salmon initially had the highest lice counts on fins, followed by Atlantic Salmon. A strong correlation was observed between mucous cell area and susceptibility, with Chum Salmon showing 30-40% mucous cell coverage compared to 10% in Atlantic Salmon. Coho Salmon successfully rejected copepodids after two days, which was linked to an influx of polymorphonuclear cells at attachment sites. This immune response, highlighted by spatial transcriptomics, showed upregulation of host immune markers at the louse-host interface, suggesting that macrophages and neutrophils are key to copepodid rejection. The findings suggest that Coho Salmon’s rapid inflammatory response is crucial in resisting lice and differs significantly from the response in Atlantic Salmon. fish skin spatial transcriptomics histopathology mucous cells L. salmonis Atlantic Salmon Chum Salmon Coho Salmon Pink Salmon Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Highlights Sedation aids louse settlement on Salmonid species with more mucous cells. Coho Salmon rejects copepodids after 2 days. Significant immune response observed in Coho Salmon towards L. salmonis . Initial louse attachment phase key to resistance. Introduction Atlantic Salmon ( Salmo salar ) is one of the most of the most valuable farmed fin fish in aquaculture, but it’s sustainable production is hampered by both the salmon louse ( Lepeophtheirus salmonis ) parasitism and treatments to remove the louse (Fast, 2014 ; Sommerset et al., 2023 ). L. salmonis exhibits a moderate level of host specificity, primarily infesting fish belonging to the Salmo and Oncorhynchus genera (Pike & Wadsworth, 1999 ). Variations in susceptibility to L. salmonis infestations have been noted between salmonid species (Johnson & Albright, 1992a ; Pike & Wadsworth, 1999 ) with the pacific species, Coho Salmon ( Oncorhynchus kisutch ), Pink Salmon ( Oncorhynchus gorbuscha ) and Chum Salmon ( Oncorhynchus keta ) regarded as more resistant towards L. salmonis compared to the highly susceptible Atlantic Salmon (Jones et al., 2007 ; Sutherland et al., 2014). The parasite has a direct life cycle including two free-swimming naupliar stages, one free-swimming infectious copepodid stage, two attached chalimus stages, two preadult stages and an adult stage (Hamre et al., 2013 ; Johnson & Albright, 1991 ). Initially the louse attaches to fin, skin or gills of the fish, and the attachment is facilitated by the free-swimming larval phase. L. salmonis feeds on fish skin, mucus and blood, and typically, L. salmonis attachment lesions are characterized by inflammatory changes ranging from ulcerative necrosis, localized hemorrhage and mixed leucocytic infiltration, with comparable responses in the species of Oncorhynchus and Salmo genera (Fast et al., 2002 ; Jones et al., 2007 ). In Atlantic salmon, L. salmonis infestation triggers a rapid and large-scale cellular immune response (Krasnov et al., 2012 ; Skugor et al., 2008 ; Tadiso et al., 2011). The T-cell response is thought to dominate in louse-infested Atlantic Salmon skin (Fast et al., 2006 ; Skugor et al., 2008 ), a hypothesis recently supported by single-nuclei RNA sequencing (snRNAseq). Despite this, the acute inflammatory immune response in Atlantic Salmon is ineffective, potentially due to the immunosuppressive nature of L. salmonis , and rejection does not occur (Braden et al., 2020 ; Tully & Nolan, 2002). Although various molecules produced by L. salmonis have been shown to possess immunomodulatory properties (Fast et al., 2007 ; Midtbø et al., 2024 ; Øvergård et al., 2022 ), these analyses have primarily provided targeted data regarding the host-parasite interaction. Moreover, the localized transcriptomic response at the specific point of louse attachment, including differences between epidermal and dermal tissues (Sveen et al., 2021a), is distinct from the broader immune response to the parasite (Øvergård et al., 2023). New methods such as spatial transcriptomics, which covers the transcriptome at spatial resolution, offers a platform to unravel the more complex picture of louse-host pathobiology (Robinson et al., 2022 ). With protocols available for fish skin (Sveen et al., 2023), this approach represents valuable tool for understanding the underlying mechanisms of hos parasite interactions. Unlike Atlantic salmon, Coho Salmon reject L. salmonis during initial phases of infestation, believed to result from non-specific host responses, including dermal inflammation as early as 1 day post-infestation (Johnson & Albright, 1992b ), chitin sensing, and immune signaling (Braden et al., 2023 ). Additionally, cortisol, known for its immune-suppressive effects, increases the susceptibility of Coho Salmon to L. salmonis infestation when cortisol implants are used (Johnson & Albright, 1992a ). Our recent snRNA-seq study further suggests that macrophages may play a key role in the Coho Salmon’s immune response towards L. salmonis (Salisbury et al., 2024 ). Moreover, Braden et al. ( 2023 ) reported elevated expression of clec4e in Coho Salmon 4–7 days post-infestation with L. salmonis . Since clec4e is a marker for neutrophils in Coho Salmon (Salisbury et al., 2024 ), this finding indicates an increase in neutrophil infiltration at the louse attachment sites. Another proposed mechanism for parasite resistance in Coho Salmon involves hyperplasia of epithelial cells, specifically keratinocytes, along with encapsulation (Braden et al., 2023 ; Johnson & Albright, 1992a ). However, findings on parasite resistance in Coho Salmon remain somewhat inconclusive, and overall, a similar immune response appears to be activated in both Atlantic Salmon and Coho Salmon skin and fin during lice infestation (Salisbury et al., 2024 ). The aim of this study was to identify early resistance mechanisms against L. salmonis in four salmonid species: Pink Salmon, Coho Salmon, Chum Salmon, and Atlantic Salmon. Samples were collected at four time points within the first 2.5 days post-infestation (dpi): 12-, 24-, 36-, and 48-hours post-infestation (hpi). Due to low attachment success in Pink and Chum Salmon, our focus was directed towards the louse attachment sites on the fins of Atlantic and Coho Salmon. Using a combination of histological techniques, including spatial transcriptomics, we show that the first 48 hpi are critical for copepodid rejection in Coho Salmon, with L. salmonis being expelled through an acute inflammatory response. These findings highlight the species-specific immune mechanisms underlying early host resistance to parasite infestation. Material and Methods 1.1 Source of fish Coho Salmon (1–2 g) and Pink Salmon (0.25–0.3 g) were transferred from Quinsam River Hatchery, Nanaimo, British Columbia, and Chum Salmon were transferred at ~ 0.6 g from Inch Creek Hatchery, Dewdney, British Columbia, to the same Level III Quarantine Facility (CATC), Prince Edward Island, Canada (March 2021). This involved packaging in chilled coolers containing freshwater from the source, supersaturated with oxygen (ca. 250%), and a transport density of 75–100 g / (30 X 15 X 16-inch shipping boxes), overnight (< 36 hr transit time). Two thousand Atlantic Salmon eggs from 10 families were donated to the project by StofnFiskur HF (Benchmark, Iceland) and synchronized to 360 °d at delivery and shipped chilled on ice to The Center for Aquaculture Technologies Souris Canada (CATC) in late November 2020. 1.2 Animal husbandry Fish were acclimated to ambient recirculating conditions (freshwater, 10–12°C) over several hours in 150 L tanks. All four species were maintained under these conditions (12 h light: 12 h dark, 2–4 L / min flow, 90–110% DO, and fed 2–3 times per day to satiation) until they reached the target weight for the lice challenge test experiment (ca. 25 g). When the population average weight reached 15 g, along with visual indicators of smoltification such as loss of parr marks, Instant Ocean® (Instant Ocean, Blacksburg, USA) was slowly added to the partially recirculating system such that salinity was increased by 4–5 ppt per day to achieve 32–33 ppt in 7 days. Fish were fed to satiation 2–3 times daily with a commercially available diet (Skretting; Skretting, #2 Crum initially and later 2.3 Nutra RC). In total 600 naïve fish, 150 of each salmonid species: Atlantic, Coho, Pink and Chum Salmon, were incorporated into a lice challenge trial at CATC, Canada. On the day of distribution to the study tanks, bulk weights of a minimum of 50 individuals per species were recorded (Average size: Atlantic salmon 40.0 g; Coho 12.5 g, Pink 50.1 g, Chum 80.6 g). All fish were in good health prior to being enrolled in the study. The experimental setup involved tanks that were supplied with recirculating aquaculture system (RAS) saltwater at a concentration of 32–37 ppt. The holding temperature was maintained at 12 ± 1°C throughout the study. A total of 20 tanks with a combined capacity of 135 L were used. Fish of each of the salmonid species were distributed over 5 tanks (3 tanks with infestation and 2 control tanks), giving 30 fish per replicate tank and 150 fish in total per species. For the experimental tanks, the flow rate of the water within the tanks was maintained at around 2–4 liters per minute, ensuring approximately one tank exchange per hour. Dissolved oxygen saturation levels were maintained between 90–120% to ensure adequate oxygenation for the fish. A standard photoperiod of 12 hours of light followed by 12 hours of darkness was implemented to simulate natural light conditions. This light cycle was regulated throughout the study. Fish were fed to satiation once daily with commercial pelleted diet appropriate for the fish size throughout the study period. Salinity, temperature, and dissolved oxygen were maintained within specified ranges. Fish were monitored for mortality and / or signs of morbidity twice daily as a standard husbandry procedure. Before the final challenge (challenge 3), fish were transferred from the RAS to a flow through system. However, tank size, water flow and light intensity remained identical to that of the RAS. 1.3 Origin and cultivation of L. salmonis Egg strings from adult female lice ( L. salmonis ) were collected from Atlantic salmon farms located in the Bay of Fundy, Atlantic Canada. Copepodids were generated by incubating the strings in a static laboratory seawater system. Concentration and viability of copepodids were validated under a stereo light microscope immediately before each challenge. The copepodids were aliquoted for challenge exposure in each individual tank. The challenge duration and concentration of copepodids per fish was adjusted depending on the challenge test methodology (Table 1 ). Table 1 Overview of the challenge experiments . Fish species, number of fish, challenge method, exposure duration and L. salmonis concentration during each challenge. Date Fish species #Fish challenged + # Tanks Challenge method Challenge duration L. salmonis concentration (#/fish) Challenge 1 13th of Oct 2021 Atlantic 90 3 Static in 100L container * 3 hours 82.2 Chum 90 3 Coho 90 3 Pink 90 3 Challenge 2 16th of Oct 2021 Atlantic 75 3 Static in tanks 2 hours ˟ 149.3 Chum 75 3 Coho 75 3 Pink 75 3 Challenge 3 26th of Oct 2021 Atlantic 66 3 Static in 10L recovery bath ‡ 15 minutes 49.8 Chum 66 3 Coho 66 3 Pink 66 3 + Fish were equally distributed into challenge tanks that served as replicates. Additional control tanks without any lice exposure were utilized for every species at each challenge. * Fish were netted out and challenged outside the tank in a 100L container. After challenge, fish were netted back to its respective tank. ‡ Fish were netted out, anesthetized, and recovered in 10L bath with lice. Post challenge entire content of a bucket including fish, lice, and water were added back to respective tank. ˟ Water supply resumed after 3 hours of static challenge. 1.4 Challenge experiments In total three consecutive immersion challenge trials on the same batch of fish were performed (Table 1 ), and lice production and challenge methodologies used a combination of those described by (Jones et al., 2007 ; Poley et al., 2018 ). Briefly, in the first challenge, 12 tanks (30 fish / tank) were infested with L. salmonis copepodids. Prior to exposure, the fish were netted from their original tanks and equally distributed into 100 L challenge containers that served as replicates. The fish were challenged with 82.2 copepodids / fish in static 100 L containers for 3 h. Additional control tanks without any lice exposure were utilized for every species at each challenge. Supplemental oxygen was added during the infestation procedure to maintain 6.0–9.0 mg / L O 2 for the 3 h exposure period. After the 3 h exposure, fish were transferred to their original fish tanks. Since the first challenge did not produce sufficient infestation levels in Chum Salmon Chum and Pink Salmon, a second challenge was carried out. This challenge was similar to the first challenge, but with reduced fish number (25 fish / tank), due to sampling a subset of fish each time to examine challenge success and a higher lice concentration, 149.3 lice / fish for 2 h. Again, a low number of lice were observed in Chum and Pink Salmon. In both the first and second challenge, Pink and Chum Salmon exhibited hyperactive swimming activity. Due to low numbers of lice in the first two challenges the exposure method was modified for the 3rd challenge. During the 3rd challenge each tank of fish was anesthetized in TMS (100 mg / L) and the anesthetized fish recovered in a static system containing lice. In total, 22 fish / tank were netted out, anesthetized, and recovered in 10 L baths with L. salmonis at a concentration 49.8 lice / fish for 15 min. Dissolved oxygen was supplemented using air-stones during the lice challenge tests. Post challenge the entire content of the 10 L container, including fish, lice, and water were added back to the respective tank. To maintain an adequate number of challenged fish, in addition to the original lice exposure tanks, one control tank was utilized in Challenge 3. For each challenge, control tanks contained the same number of fish as the lice exposure tanks for each species. Control fish were handled the same as the challenged fish in every possible way except being exposed to the lice. 1.5 Lice counting and tissue sampling Tissue sampling and lice counts were performed at six time points post infestation, 12 hpi, 24 hpi, 36 hpi, 48 hpi, 60 hpi and 168 hpi, while naïve control samples were collected at 36 and 168 hpi (Fig. 1 ). For tissue sampling, fish (n = 3 / tank; n = 9 per species per time point) were mildly sedated using an anesthetic dose (100 mg / L) of TMS (Syndel, Nanaimo, BC, Canada) and then were administered a lethal blow to the head. Individual fish were placed in a separate container so that lice were not lost during processing. Fish were processed prior to lice counts by bleeding, and each opercula, gill arch, and fin was placed in a petri dish containing sea water. Finally, attached L. salmonis were counted by life stage and tissue type (body, fins, or gills) using a stereomicroscope. After the final challenge, the study continued until 168 hpi, at which point all remaining fish (n = 7 per tank) were sampled as above. Skin was collected from a standardized position on the flank of the fish (Fig. 1 ) and transferred to 10% buffered formalin (Fisher-Scientific, Missassauga, ON, Canada) for histological analysis. Fins with lice attached were transferred to 10% buffered formalin (Fisher-Scientific, Missassauga, ON, Canada), or embedded in O.C.T Embedding Matrix (CellPath, UK) as described by Sveen et al 2023. A full list of lice counts, and samples collected during the challenge experiments, including those for genetic analysis, may be found in Supplementary file 1. Samples for histological analysis and spatial transcriptomics were shipped from Canada to Norway using Cencora World Currier™ with temperature loggers. 1.6 Processing of tissue samples for histological examination Embedding, sectioning, and staining of the tissue samples (skin and fin) was done at the Veterinary Institute in Harstad, Norway. For the fin samples with L. salmonis attached the samples were trimmed under a stereoscope at the position of the louse (2 x 3 mm) prior to embedding the samples. Tissue samples with registered louse at sampling, but no visual observation of the louse during examination in the laboratory were excluded from the analysis, resulting in a total of 15 samples with attached louse in Atlantic Salmon and Coho Salmon (Table 2 ). Paraffin embedded tissue sections were hydrated in water and stained with 1% Alcian blue (AB, Alfa Aesar) in 3% acetic acid for 15 min, transferred to 1% periodic acid (VWR) for 10 min, followed by Schiffs (Sigma-Aldrich®, Saint-Louis, MO, USA) reagent for 15 min, and finally for 30 s in hematoxylin (VWR, Radnor, PA, USA) before dehydration, mounting and AB / PAS (Alcian Blue / Periodic acid – Schiffs) staining (which stains mucous cells blue, purple or pink based on the acidity of the mucins). MOVAT staining was carried out on one sample (Coho Salmon) with lice attached, flowing the protocol outlined by (Suvarna et al., 2018). The stained tissue sections were scanned at 20 X with Hammamatsu Nanozoomer S360 and uploaded in the Aiforia® software for visualization. Selected sections were viewed and photographed at higher magnifications (40 X, 60 X and 100 X) with Zeiss Axio Observer Z1 (Zeiss, Oberkochen, Germany). The sections were analyzed by a trained histologist. Multiple parallel sections were done for each sample, leaving no extra sample material for additional analysis. Table 2 Fin samples with L. salmonis attached processed for histological analysis. The sampled lice attachment site varied, and is denoted as body flank (BF), caudal fin (C), pectoral fin (PC), adipose fin (A) or dorsal fin (D) Atlantic salmon hpi 12 12 12 24 24 24 36 36 36 48 48 60 168 168 168 Body site PC C BF BF BF BF BF BF A PC C PC BF BF C Coho Salmon hpi 12 12 12 24 36 36 36 48 48 48 48 48 48 48 168 Body site D D PC C C PC C C PC PC PC D C AD C 1.7 Immunohistochemistry Immunohistochemistry (IHC) with primary antibodies for proliferating cell nuclear antigen (PCNA) and Tumor Necrosis Factor Alpha (TNF-α) (Vertebrate Antibodies, UK) was carried out on a total of six selected samples from Coho Salmon, three samples for each primary antibody. In brief, immunohistochemistry (IHC) was performed by blocking de-paraffinized tissue sections with horse serum (Sigma-Aldrich, Missouri, USA) for 20 min, followed by primary antibody, being either proliferation nuclear cell antigen (dilution 1 : 60), or TNF-α (1 : 2000). Slides were washed twice in PBS before incubating with ImmPress AP-anti-Rabbit IgG for TNF-α (Vector Laboratories) or AP-anti-mouse IgG for TNF-α (Vector Laboratories) for 30 min. Immpact Vector Red substrate kit (Vector Laboratories) with alkaline phosphatase was used for 30 min according to the protocol before counterstaining 30 s with haematoxylin (Sigma). Slides were visualized as described in the previous section. 1.8 AI – analysis of skin from body flank Skin samples from the body flank were analyzed using Nofima’s skin-AI model on the Aiforia® platform, as outlined by (Sveen et al., 2021b). The analysis included tissue samples from the following four species and time points: Atlantic Salmon (N = 6 control, N = 9 infested (4 samples 12 hpi, 5 samples 36 hpi)), Chum Salmon (N = 10 control, N = 14 infested (8 samples 12 hpi and 6 samples 36 hpi)), Coho Salmon (N = 9 control, N = 13 infested (6 samples 12 hpi, 7 samples 36 hpi)), Pink Salmon (N = 10 control, N = 15 infested (7 samples 12 hpi, 8 samples 36 hpi)). Post-AI-analysis, the results were exported, filtered as described by Sveen et al. 2021b, and further analyzed in R. Given no significant differences in skin parameters between the infestation at the two timepoints (12 hpi and 36 hpi), these were combined for the final analysis, comparing infestation and species. Statistical calculations included two-way ANOVAs, Tukey post-hoc tests, and Pearson correlations, using the provided stats package functions (aov(), TukeyHSD(), and cor.test()). The results were visualized using a combination of custom functions and the beeswarm package. 1.9 Spatial transcriptomic of lice attachment site in Coho Salmon In total four samples, two Coho Salmon fins, and two Atlantic salmon fins with L. salmonis attached were processed for spatial transcriptomics, as described by (Sveen et al., 2023). Libraries were sequenced on NovaSeq 6000 SP flow cell (Illumina, USA) at the Norwegian Sequencing Center as 50 bp paired end reads. Sequencing was done using the following cycles: read 1, 28 cycles, i7 Index; 10 cycles, i5 Index; 10 cycles; and read 2, 90 cycles (Visium Spatial Gene Expression User Guide; 10x Genomics). Reads were aligned to the Atlantic Salmon genome (version Ssal_v3.1, INSDC Assembly GCA_905237065.2), Coho Salmon Genome build Okis_V2, accession number GCA_002021735.2 and L. salmonis Genome build UVic_Lsal_1.0, accession number GCA_016086655.3 () using the software Space Ranger (version 1.3.1; 10x Genomics, USA). Sequence data have been submitted to NCBI's Sequence Read Archive (SRA) as BioProject PRJNA1149653. High-resolution JPG images (Supplementary file 2–5) from each of the associated tissue sections were aligned to the reads by default settings. Optimal number of tissue clusters and cluster membership of spots was defined using graph-based clustering [modified python implementation of the augmented implicitly restarted Lanczos bidiagonalization algorithm (IRLBA) (Baglama and Reichel 2005)] in Space Ranger. Transcripts defining tissue clusters were filtered using the following criteria: only upregulated transcripts (relative to the other clusters), adjusted P-value (Benjamini–Hochberg procedure) 1. Expression levels of genes were visualized using 10x Genomics Loup Browser v8.0.0. Fine tuning of clustering was done using k-means in 10x Genomics Loup Browser v8.0.0. Searching for differentially expressed genes at the host parasite interphase using k-mean function. For L. salmonis K-mean were set to 5 and differentially expression ran excluding cluster 1. Further, top 100 DEG for cluster 3, 4 and 5 (host / parasite interface), were exported followed by manual curation for genes involved in immune function / regulation. Results and discussion 2.1 L. salmonis count After the first two challenge exposures there were no copepodids found on any Pink or Chum Salmon and only a limited number of copepodids found on Atlantic salmon (mean = 8.0 +/- 6.4 SD) and Coho Salmon (mean = 1.0 +/- 1.0 SD). These infestations did not progress past 8 days. In the third challenge, the total average lice count on Atlantic Salmon was 13.44 / fish (SD 4.52) at 12 hpi, while total average counts in Coho, Pink, and Chum Salmon were 3.89 / fish (SD 1.79), 2.89 / fish (SD 4.31), and 5 / fish (SD 2.05) respectively (Fig. 2 ). Total counts in Pink and Chum Salmon decreased to 0.44 (SD 0.96) and 1.40 (SD 1.08) respectively at 24 hpi, and both species dropped to 0 lice / fish by day 7 (termination). Most of the settled lice on Atlantic Salmon progressively moulted to later developmental stages, e.g. chalimus at 168 hpi. At termination, the total average lice count on Coho and Atlantic Salmon were 0.62 and 11.24 lice / fish respectively. Notably, lice count on Coho fins was initially higher than on any of the other salmonid species but was only sustained for 48 hpi before decreasing to 0.57 lice on fins per fish at termination. 2.2 Quantitative assessment of fish skin Quantitative histological analysis of skin from the body flank of the fish showed no significant differences in the epithelial tissue or mucous cell number between infested and control samples within each salmonid species (Fig. 3 ). However, there were differences between the species. Chum Salmon had the largest area of epithelial tissue per mm of skin, and 30–40% of the epithelial area was mucous cells, corresponding to more than 100 mucous cells per mm of skin. Additionally, mucous cells accounted for 15–20% of the epithelial surface in Pink and Coho Salmon, while Atlantic Salmon showed the lowest proportion, with only 10% coverage. There is ongoing debate in the literature regarding which salmonid species is most resistant to salmon lice infestation. Experimental trials with L. salmonis have shown that Chum Salmon are more susceptible to lice compared to Pink Salmon (Jones et al., 2007 ) and Atlantic Salmon (Sutherland et al., 2014). However, another study found that wild Pink Salmon exhibited a higher lice prevalence compared to Chum Salmon (Nagasawa, 2001 ). Notably, under experimental conditions, both Chum Salmon and Pink Salmon must be sedated to facilitate infestation. Hence, based on our findings, we suggest that mucous cells, potentially in combination with behavioral mechanisms (disrupted by sedation), may offer protection against lice settlement in Chum and Pink Salmon. Additionally, factors beyond mucous cell count, such as variations in presence of immunological defense molecules (Esteban, 2012 ), or organic compounds (Difford et al., 2022 ) in the fish mucus, could contribute to L. salmonis resistance and warrant further investigation. 2.3 Assessment of the attachment site of L. salmonis Given that L. salmonis successfully attached to the fins of both Atlantic Salmon and Coho Salmon, but only Coho Salmon displayed copepodid rejection, we directed our investigation towards a more detailed analysis of the attachment sites in these two species. In Atlantic Salmon signs of tissue damage inflicted by lice were apparent as early as 12 hpi, with epithelial tissue erosion and a reduction in mucous cells directly beneath the mouth tube of L. salmonis (Fig. 4 ). Concurrently, an influx of polymorphonuclear cells was observed within dermal blood vessels beneath the attachment site. Over later time intervals (36–168 hpi), there were no polymorphonuclear cells directly at the feeding site. This suggests that the inflammatory response at later stages may have been reduced by the louse’s immunomodulatory effects (Braden et al., 2020 ; Hamilton et al., 2018 ; Midtbø et al., 2024 ; Øvergård et al., 2022 ). Further, lice induced tissue damage included the depletion of mucous cells beneath the louse and erosion of the epithelium (Fig. 5 ), aligning with previous research (Tully & Nolan, 2002). Although most samples showed a decrease in mucous cell abundance at the feeding site, some exceptions were noted. Notably, two Atlantic Salmon samples had a significant increase in purple-staining mucous cells at the louse attachment site, with similar observations in Coho Salmon (Fig. 6 , Supplementary file 1). In healthy Atlantic salmon skin, purple mucous cells are typically sparsely distributed (Sveen et al., 2021b), but lice infestation may increase in their prevalence at attachment site (Sveen et al., 2021a). The variation in mucous cell color holds significance for mucus function, indicating a glycosylation shift in mucin proteins (Zaretsky & Wreschner, 2013). Transitioning from blue-staining to purple-staining mucous cells, with Alcian blue and PAS staining, suggests alterations in glycans on the mucin backbone, potentially resulting in changes to mucus pH, solubility, and its capacity to interact with and bind to pathogens, such as bacteria (Padra et al., 2014 ; Padra et al., 2017 ; Padra et al., 2019 ). Moreover, several pathogens, both bacteria and parasites, can employ a strategy known as "glycan gimmickry," wherein they produce glycans that mimic those of the host. This mimicry can facilitate interaction with host lectins, potentially modulating or evading immune responses (Rodrigues et al., 2015 ). Therefore, the dynamics of the mucous cell population and mucin glycan pattern could shed light on mechanisms underlying host defense evasion and potentially inform novel intervention strategies. Coho Salmon demonstrated a heightened immune response towards L. salmonis , particularly between 36 and 48 hpi, which corresponded to the time point before the observed reduction in lice numbers. Among the ten samples analyzed in this time interval, four exhibited a robust influx of polymorphonuclear cells not only within blood vessels but also infiltrating the dermal tissue (Fig. 7 ). Remarkably, in three of these samples, polymorphonuclear cells filled the entire mesenchymal cavity of the fin contrasting sharply with the inflammatory reaction observed in Atlantic Salmon. Neutrophils were initially identified as the predominant cell type at the site of louse attachment in both Atlantic Salmon and Coho Salmon through histological techniques (Johnson & Albright, 1992a ), however precise classification of immune cells by visual examination can be difficult. Analysis of raw and normalized snRNA-seq counts for inflammatory cells at the attachment site in fin and skin samples (Supplementary file 6; data from Salisbury et al., 2024 ) show an increased proportion of both neutrophils and macrophages at 12 hpi in Coho Salmon fin samples. This increase was not observed in Coho Salmon skin, Atlantic Salmon fin, or Atlantic Salmon skin samples. Additionally, samples with significant infiltration of polymorphonuclear cells also showed positive staining for anti-TNFα (Fig. 8 ), a cytokine predominantly produced and released by activated macrophages that plays a central role in recruiting leukocytes to inflamed tissues. In one sample, the cephalon (head) of the louse was precisely captured at the attachment site, offering insights into how Coho Salmon may reject or combat L. salmonis infestations at the copepodid stage (Fig. 9 ). Notably, polymorphonuclear cells were observed in the epithelial compartment near the L. salmonis head and were abundant in the dermal compartments directly beneath the louse. The characteristics of these cells varied depending on their location; those closest to the louse exhibited fragmented nuclei within a single cell body (Supplementary file 7), which may indicate they are undergoing apoptosis, a common feature of activated immune cells (Donovan et al., 2021 ; Ekert & Vaux, 1997 ). Moreover, significant alterations in the epithelial tissue and basement membrane were observed at the louse attachment site, highlighted by red staining with MOVAT pentachrome (Fig. 9 ). In contrast, both the basement membrane and epithelium retained normal staining at increasing distances from the L. salmonis head. This red staining may indicate fibrinoid necrosis (Movat, 1960 ), a specific type of tissue necrosis characterized by the deposition of fibrin-like proteinaceous material, commonly associated with immune complex-mediated diseases and inflammatory responses (Bajema & Bruijn, 2000 ; Movat & More, 1957 ). Previous studies on louse-infested Coho Salmon have also reported necrotic tissue at louse attachment sites (Johnson & Albright, 1992b ). Additionally, this region displayed signs of hyalinization, edema and epithelial vacuolization, indicating that the strong inflammatory response leads to tissue damage. The extent of this damage suggests that active degranulation may be occurring (Hirsch & Cohn, 1960 ). Degranulation typically involves the release of pro-inflammatory factors, such as histamines, leukotrienes, and cytokines (Gierlikowska et al., 2021 ), which target invading pathogens but can also cause collateral damage to host tissues. Following degranulation, immune cells often undergo apoptosis to limit excessive tissue damage and inflammation (Soehnlein et al., 2008 ). An apoptotic process will affect the cell nuclei, and may partly explain why granulocytes were difficult to detect in our snRNAseq analysis (Salisbury et al., 2024 ). Finally, immunohistochemistry with PCNA (a marker for cell proliferation) was performed on two samples of Coho Salmon: one featuring the captured head of the louse, and the other exhibiting notable epithelial tissue hyperplasia towards L. salmonis (Fig. 10 ). PCNA + cells were predominantly observed in the sample displaying hyperplasia, but not in the sample with abundance of polymorph nuclear cells. Localized hyperplasia of the epithelial tissue and keratinocyte proliferation, occasionally leading to the encapsulation of entire lice, has been generally recognized as a pivotal factor driving lice resistance in Coho Salmon (Johnson & Albright, 1992a ; Salisbury et al., 2024 ). However, within the timeframe of 36 to 48 hours post-infestation in the present study, only one sample exhibited this characteristic. This may suggest that it is the acute inflammatory response, and not epithelial hyperplasia, is the primary mechanism acting against copepodids in Coho Salmon. 2.4 Transcriptomic responses at the intersection of louse and host To further characterize the host responses to L. salmonis , we employed spatial transcriptomics on Atlantic and Coho Salmon fins with L. salmonis attached in the first hours after infestation. We were only able to detect tissue from L. salmonis and salmon tissue on one slide, mainly due to the technical difficulty of pinpointing the exact point of attachment between the host tissue and the small L. salmonis copepodid which is less than 1 mm in size at the copepodid stage. Further, given that differentially expressed genes are determined within each tissue slide rather than across multiple samples, we directed our analysis towards the specimen containing both louse and host tissue and manually curated for annotated genes that were DEGs at the intersection between the host and the parasite. This analysis was performed separately for the Coho Salmon genome and the L. salmonis genome. 2.4.1 Spatial transcriptomic slide mapped to the genome of Coho Salmon Tissue ( L. salmonis + Coho Salmon) covered 250 bar coded spots. For the Coho Salmon total genes detected were 18218 and the median genes per spot were 1252. The UMI counts overlapped with both the Coho Salmon tissue and L. salmonis tissue, however with lowest count on the L. salmonis tissue (Fig. 11 A). This overlap could potentially be due to the feeding behavior of L. salmonis on host tissue, including consumption of immune cells at the point of attachment. Manual curation for differentially expressed genes at the attachment site resulted in 17 genes showing high specificity at the host-parasite interface (Table 3 ), with eight of these being most specific to the area of attachment (Fig. 11 B and C). The differentially expressed genes at the attachment site were associated with immune responses, oxidative stress responses, proteolysis, and cell migration, all characteristic of acute inflammation (Soliman & Barreda, 2022 ). Genes involved in immune responses and proteolysis included granulocyte colony-stimulating factor receptor ( csf3r ), myeloperoxidase ( mpx ), lysozyme ( lyz ), matrix metalloproteinase 9 ( mmp9 ), cathepsins ( ctss ), metalloproteinase inhibitor 2-like ( timp2 ), and arachidonate 5-lipoxygenase ( alox5a ). Notably, myeloperoxidase , an enzyme critical for the antimicrobial activity of neutrophils and granulocyte colony-stimulating factor receptor , a receptor that stimulates neutrophil production, were highlighted. Furthermore, inflammatory cells, particularly neutrophils and macrophages, are known to have high concentrations of proteases, including lysozyme, matrix metalloproteinases, and cathepsins (Soliman & Barreda, 2022 ), hence the upregulation of metalloproteinase inhibitor 2-like is likely to counterbalance protease activity. Further, the production of reactive oxygen species (ROS) is essential for antimicrobial activity and immune signaling. Genes such as glutathione peroxidase 1b ( GPX1b ) and metallothionein suggest active antioxidant defenses at the attachment site, providing protection against ROS. Additionally, the expression of thymosin beta 4 and junB proto-oncogene , which are involved in various cellular processes including cell migration, angiogenesis, and wound healing (Sveen et al., 2019; Sveen et al., 2018), suggests activation of cell migration. Finally, in our recent single-cell study, mmp9 was identified as a marker of neutrophils in Atlantic Salmon, while csf3r (ENSOKIG00005018820) was found to be highly expressed in both neutrophils and macrophages (Salisbury et al., 2024 ). A different paralogue of ctss (i.e., ENSOKIG00005022279) than the one detected here is also known as a macrophage marker (Salisbury et al., 2024 ). Hence, the upregulation of these genes, therefore, supports the infiltration of macrophages and neutrophils to the attachment site of L. salmonis in Coho Salmon. Table 3 Differentially expressed genes at L. salmonis attachment site in Coho Salmon. Gene ID Symbol Name ENSOKIG00005043004 alox5ap arachidonate 5-lipoxygenase-activating protein-like ENSOKIG00005021726 ctss cathepsin S-like ENSOKIG00005007964 coro1a coronin-1A-like ENSOKIG00005049844 mpx eosinophil peroxidase ENSOKIG00005011185 fbp1 fructose-1,6-bisphosphatase 1 ENSOKIG00005040066 Gpia glucose-6-phosphate isomerase ENSOKIG00005014626 gpx1b glutathione peroxidase 1b ENSOKIG00005018820 csf3r granulocyte colony-stimulating factor receptor ENSOKIG00005018202 junb JunB proto-oncogene, AP-1 transcription factor subunit ENSOKIG00005050890 lyz lysozyme [Source:NCBI gene ENSOKIG00005010179 mmp9 matrix metalloproteinase-9 ENSOKIG00005043531 mmp9 matrix metalloproteinase-9-like ENSOKIG00005030083 mt2 metallothionein B ENSOKIG00005046918 nccrp1 NCCRP1, F-box associated domain containing ENSOKIG00005018695 oscp1 protein OSCP1 ENSOKIG00005035017 timp2b metalloproteinase inhibitor 2-like ENSOKIG00005025750 txnip thioredoxin-interacting protein-like 2.4.2 Spatial transcriptomic slide mapped to the L. salmonis genome A total of 8885 genes were detected for L. salmonis . The louse tissue covered only 12 barcoded spots on the spatial transcriptomics slide, corresponding to the approximate size of the parasite (500 µm). The UMI counts mapped to these 12 spots ranged from 8000 to 34372 (Fig. 12 A and B). Analysis of differentially expressed genes (DEGs) at the host-parasite interface, particularly those related to regulation and immunity, identified 14 louse genes with a fold change greater than 4 (Table 4 ). While some of these genes are directly associated with immune responses (e.g., tlr7 , notch1b ), others may influence immune-related pathways indirectly or be involved in broader regulatory processes within the parasite. The upregulation of these genes could indicate adaptations of the parasite to its host environment, including mechanisms to evade host immune responses or modulate host cell functions (Øvergård et al., 2022 ). However, given that only a single specimen was analyzed and the L. salmonis transcriptome remains difficult to interpret due to a poorly annotated genome (with over 13.000 protein-coding genes, many of which are uncharacterized), we are cautious in drawing definitive conclusions. Nevertheless, this study demonstrates the feasibility of capturing the transcriptomes of two different species on a single spatial transcriptomics slide. With further refinement, this methodology could provide novel insights into co-regulation and cross-species signaling mechanisms. Table 4: Differentially expressed L. salmonis genes at the attachment Site. Cluster 2 represents 12 barcoded spots corresponding to L. salmonis tissue. Clusters 3, 4, and 5 each consist of a single barcoded spot located at the host-parasite interface (refer to Figure 12). Red shading indicates positive fold changes, while blue indicates negative fold changes relative to other clusters. 2.5 Concluding remarks The observed infiltration of polymorphonuclear cells at attachment sites in Coho Salmon, coupled with the upregulation of genes related to immunity at the direct interface between the host and parasite, indicates a concerted effort by the immune system to combat the invading parasite. However, it's important to acknowledge that this study is based on a limited number of samples, primarily due to challenges in obtaining high-quality sections from verified louse attachment sites. Despite the relatively small sample size, compelling evidence supports an acute inflammatory response characterized by the recruitment of neutrophils and macrophages, leading to the effective expulsion of copepodids in Coho Salmon. Moreover, Coho Salmon likely employs multiple resistance strategies, combining a strong inflammatory response against copepodids with epithelial hyperplasia, which may become more important when copepodid rejection is incomplete (Fig. 13 ). When considering the challenge of developing resistance, it’s important to recognize that overcoming two different genetic resistance mechanisms requires at least two types of adaptations in the pathogen. A single variant or mutation is insufficient for the louse to survive and reproduce successfully. This means that if Coho Salmon possess multiple resistance mechanisms against the louse, it will take significantly longer and be much more difficult for the louse to develop resistance. This could explain why we haven’t observed resistance development in L. salmonis towards Coho Salmon. This study demonstrates that acute inflammation targeting the copepodid stage is likely the primary mechanism driving salmon lice resistance in Coho Salmon. Moreover, some of the identified genes and pathways at the attachment site in Coho Salmon could be promising candidates for gene editing or modulation to assess their effects on L. salmonis infestation in Atlantic Salmon. Declarations Ethical statement All fish handling and procedures were approved by CATC and UPEI Animal Care Committees (AUP 21-008) and carried out under the direct supervision of trained scientists in strict compliance with regulations set out by the Canadian Council for Animal Care (http://www.ccac.ca/). All experiments were performed in accordance with relevant guidelines and regulations, and followed recommendations described by the ARRIVE guidelines for conducting research on animals. Funding information This work was part of the “CrispResist” project funded by the Norwegian Seafood Research Fund (FHF project 901631). DR acknowledges BBSRC Institute Strategic Grants to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A) JEB acknowledges BBSRC Grant to the University of Stirling, BB/V009990/1. Conflict of Interests The authors declare that they have no conflict of interest. Acknowledgements The authors would like to thank the supply of fish (Coho and Pink Salmon) from Quinsam River Hatchery (Paige Ackerman, Edward Walls, Sherri Higgins, Cindy Ederis); Chum Salmon Inch Creek Hatchery (James Wegers); and Ólafur H. Kristjánsson of StofnFiskur, Benchmark Genetics in Iceland and Morten Rye of Benchmark Genetics in Norway for donating and organising the collection and shipment of Atlantic salmon eggs to Canada. 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Supplementary Files Supplementaryfile1.xlsx Supplementaryfile2Frame1Coho.jpg Supplementaryfile3Frame2Coho.jpg Supplementaryfile4Frame3Atlantic.jpg Supplementaryfile5Frame4Atlantic.jpg Supplementaryfile6.tif Supplementartyfile7.tif Cite Share Download PDF Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Cell and Tissue Research → Version 1 posted Editorial decision: Revision requested 13 Nov, 2024 Reviews received at journal 12 Nov, 2024 Reviewers agreed at journal 02 Nov, 2024 Reviewers agreed at journal 01 Nov, 2024 Reviews received at journal 31 Oct, 2024 Reviewers agreed at journal 29 Oct, 2024 Reviews received at journal 29 Oct, 2024 Reviewers agreed at journal 21 Oct, 2024 Reviewers agreed at journal 17 Oct, 2024 Reviewers invited by journal 17 Sep, 2024 Editor assigned by journal 21 Aug, 2024 Submission checks completed at journal 21 Aug, 2024 First submitted to journal 20 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Mohammed","email":"","orcid":"","institution":"Onda (Formerly Centre for Aquaculture Technologies Canada – CATC)","correspondingAuthor":false,"prefix":"","firstName":"Haitham","middleName":"H.","lastName":"Mohammed","suffix":""},{"id":357381906,"identity":"d84e2f0b-ba31-4136-a8c3-eb39720cd2fc","order_by":9,"name":"Rose Ruiz Daniels","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Rose","middleName":"Ruiz","lastName":"Daniels","suffix":""},{"id":357381907,"identity":"ca555b1f-8739-471e-9561-54e1ca2415cb","order_by":10,"name":"Sarah Salisbury","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Salisbury","suffix":""},{"id":357381908,"identity":"32fbccf4-3bd0-4997-a673-6bd3cc6c3742","order_by":11,"name":"Diego Robledo","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Robledo","suffix":""},{"id":357381909,"identity":"64d7dede-51cc-42a9-862f-6099902b5f96","order_by":12,"name":"Mark Braceland","email":"","orcid":"","institution":"Onda (Formerly Centre for Aquaculture Technologies Canada – CATC)","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Braceland","suffix":""},{"id":357381910,"identity":"1ff86333-a9a8-4ec1-bf49-723348e41e95","order_by":13,"name":"Miroslava Hansen","email":"","orcid":"","institution":"Norwegian Veterinary Institute","correspondingAuthor":false,"prefix":"","firstName":"Miroslava","middleName":"","lastName":"Hansen","suffix":""},{"id":357381911,"identity":"b987b76e-29d7-424a-ae36-89a6c4c5b0f7","order_by":14,"name":"Nicholas Robinson","email":"","orcid":"","institution":"Nofima Muninbakken","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Robinson","suffix":""}],"badges":[],"createdAt":"2024-08-20 21:46:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4947433/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4947433/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00441-025-03976-0","type":"published","date":"2025-06-04T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65105604,"identity":"167b9210-85dc-48f6-8267-bc57f92d322f","added_by":"auto","created_at":"2024-09-23 16:20:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1246333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue sampling protocol for the third challenge.\u003c/strong\u003eA. Tissue samples were collected frequently within the first two and a half days, at 12-, 36-, 48-, and 60-hours post-infestation (hpi), followed by a final sampling at one week (168 hpi). \u003cem\u003eL. salmonis\u003c/em\u003e were at the copepodid stage during the first 2.5 days, transitioning to the chalimus stage by the final time point. The inset images show a copepodid (arrow) and a chalimus with associated epithelial hyperplasia (arrow) in Coho Salmon. B. At each time point, skin samples from the body flank were collected and preserved in 10 % formalin. Additionally, fins with attached lice (when available) were preserved in 10 % formalin or embedded in OCT compound and stored on dry ice. The photo shows an Atlantic Salmon smolt. C. Formalin-fixed tissue containing \u003cem\u003eL. salmonis\u003c/em\u003e was carefully dissected under a stereomicroscope, with a scalpel targeting the area near the attachment site (indicated by the white rectangle). The samples were then paraffin-embedded, and serial sections were made throughout the tissue to ensure the attachment site or adjacent areas were captured.\u003c/p\u003e","description":"","filename":"Figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/c7ce3725ff293ce46d79b04c.jpg"},{"id":65105605,"identity":"de3db862-c8b9-4b6b-913d-634215e54219","added_by":"auto","created_at":"2024-09-23 16:20:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4799224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLice counts in Atlantic, Chum, Coho and Pink Salmon.\u003c/strong\u003e A. Average lice count per fish in total (body, fins and gill). B. Average lice count on fins C. Average lice count on gill D. Average lice count on body at sampling time points 12, 24, 36, 48, 60 and 168 hours post infestation. Error bars, standard error of mean.\u003c/p\u003e","description":"","filename":"Figure22.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/5497d09e6cc9d09b588a5389.jpg"},{"id":65106458,"identity":"6b59f809-ee99-4e02-a121-83aaade24efb","added_by":"auto","created_at":"2024-09-23 16:36:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":864829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative assessment of epidermal area and mucous cells in the skin of four salmonid species. \u003c/strong\u003eA. Epidermal area. B. Ratio of mucous cell area to epidermal area. C. Number of blue-stained mucous cells per mm of skin. Two-way ANOVA p-values for species and infestation, effects are indicated at the top of each plot. Significant differences between groups were determined using Tukey’s post-hoc test at p \u0026lt; 0.05; groups not sharing a letter are significantly different. Grey dashed horizontal lines represent expected median values for the given fish size (based on Atlantic Salmon averages). Red circles highlight values that are more than two standard deviations from the adjusted median.\u003cstrong\u003e \u003c/strong\u003eD-G. Representative Alcian Blue and Periodic Acid-Schiff-stained tissue sections of skin from the body flank of each species: D. Atlantic Salmon, E. Chum Salmon, F. Coho Salmon, and G. Pink Salmon. Abbreviations: epithelium (epi), scale (sc).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/ae9074a6f7a771e871d385da.jpg"},{"id":65105620,"identity":"460b17ba-0c17-41fd-b9c1-2a1bc75d601c","added_by":"auto","created_at":"2024-09-23 16:20:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1937333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue damage caused by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eat the feeding site in Atlantic Salmon fin at 12 hpi. \u003c/strong\u003eA – E. sequential sections through the feeding site. E – H. Close-up views of the feeding site, as indicated by the rectangles in panels A–D. Tissue sections in panels G and H represent areas outside the direct contact with the louse’s mouth tube. Key anatomical features are labeled in the figure: basement membrane (bm), mouth tube (mt), epithelial loss (*), epithelium (epi), mucous cell (muc), polymorphonuclear inflammatory cells (pmn), and blood vessel (bv). Tissue sections were stained with Alcian Blue and Periodic Acid-Schiff.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/25f8576a5836518154ef7a94.jpg"},{"id":65106208,"identity":"93d91adb-a449-42f7-8767-2bf0d3fbc781","added_by":"auto","created_at":"2024-09-23 16:28:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2176181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAttachment sites on Atlantic Salmon skin and fin at 48 and 168 hours post-infestation (hpi).\u003c/strong\u003e A. Caudal fin, 48 hpi. B. Body flank, 168 hpi, showing epithelial cell ingestion (black arrow). C. Body flank, 168 hpi. D. Pelvic fin, 168 hpi. E. Control, skin from body flank. The white arrow indicates the anterior direction of \u003cem\u003eL. salmonis\u003c/em\u003e. Note the reduced presence of mucous cells in the epithelium directly under \u003cem\u003eL. salmonis\u003c/em\u003e, particularly beneath the head (telencephalon), as well as the absence of polymorphonuclear cells at the feeding site. Abbreviations: epithelium (epi), mucous cell (muc), muscle (mu), scale (sc), adipose tissue (ad). Tissue sections were stained with Alcian Blue and Periodic Acid-Schiff.\u003c/p\u003e","description":"","filename":"Figure54.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/80ffa751f58db94858f0e083.jpg"},{"id":65105614,"identity":"63de7c04-8ccb-4890-8bfb-a7cb1af99f42","added_by":"auto","created_at":"2024-09-23 16:20:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1918411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased population of purple-staining mucous cells in the fin at \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e attachment sites in Atlantic Salmon and Coho Salmon. \u003c/strong\u003eA. Atlantic Salmon. The inset shows a magnified area with a mix of blue and purple-staining mucous cells. B. Atlantic Salmon, where one side of the fin displays predominantly purple-staining mucous cells, while the opposite side shows mostly blue-staining mucous cells. C. Coho Salmon, section from an individual with a marked increase in purple-staining mucous cells. In addition to mucous cells, Coho Salmon also exhibit sacciform cells in the epithelium. Tissue sections were stained with Alcian Blue and Periodic Acid-Schiff. Abbreviations: epithelium (epi), dermis (derm), mucous cell (muc), sacciform cell (sac).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/ae08d6989e239ab606ba4cb2.jpg"},{"id":65106213,"identity":"8991eb47-d76d-4346-939b-b06c251f42ee","added_by":"auto","created_at":"2024-09-23 16:28:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":320002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue responses to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e at the attachment site in Coho Salmon at 12, 36, and 48 hours post-infesation (hpi).\u003c/strong\u003e A. Caudal fin at 12 hpi. B. Caudal fin at 36 hpi, showing epithelial tissue (epi) loss at the feeding site. C. Fin with two distinct compartments of polymorphonuclear (pmn) cells. D. Pectoral fin at 36 hpi, showing a substantial influx of polymorphonuclear (pmn) cells and a high number of purple-staining mucous (muc) cells. Pectoral fin at 48 hpi with a continued large influx of polymorphonuclear (pmn) cells. F. Control sample from the body flank. The anterior cephalon of the louse (where present) is marked by an arrow. Tissue sections were stained with Alcian Blue and Periodic Acid-Schiff. Abbreviations: epithelium (epi), mucous cell (muc), polymorphonuclear cells (pmn).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/53a8ad8392e01ab514f34318.jpg"},{"id":65106209,"identity":"09415205-1bb7-436f-89c0-776f2f1c8c8a","added_by":"auto","created_at":"2024-09-23 16:28:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1881815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProminent TNFα staining at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e attachment site in Coho Salmon. \u003c/strong\u003eA. Extensive influx of polymorphonuclear cells in the dermal tissue, together with positive staining for TNFα (red) in the dermal compartment and weaker staining in the epithelium (epi). B \u0026amp; C. Positive TNFα staining in the dermal tissue, with signs of tissue degeneration (*). The area marked by the square in panel D is magnified in panel C. D. Arrows mark polymorphonuclear cells in both the dermal and epidermal compartments, along with epithelial vacuolization. E. Positive TNFα staining in the dermal compartment, showing polymorphonuclear cells within a dermal blood vessel.\u003cstrong\u003e \u003c/strong\u003eImmunohistochemistry was performed using anti-TNFα antibodies, highlighting extracellular TNFα+ areas in red. Abbreviations: epithelium (epi), vacuolization (vac), polymorphonuclear cells (pmn).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/40db0f753229f3cf7763683d.jpg"},{"id":65106459,"identity":"522d89dc-5687-4e30-8386-d022d652a95a","added_by":"auto","created_at":"2024-09-23 16:36:12","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":715191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerial sections through the attachment site in Coho Salmon at 36 hours post-infestation (hpi). \u0026nbsp;\u003c/strong\u003eA. Tissue section immediately anterior to the \u003cem\u003eL. salmonis\u003c/em\u003eattachment site. B. Same sample as in A, showing a section directly through the head of \u003cem\u003eL. salmonis\u003c/em\u003e, which is embedded in the epithelial tissue of Coho Salmon. C. Magnified view of the \u003cem\u003eL. salmonis\u003c/em\u003e head, illustrating polymorphonuclear cells in both the dermal and epidermal compartments, along with vacuolization and hyalinization of the epithelium. This image is from the same section as B, mirrored due to imaging with the Zeiss AXIO Observer at 60x magnification. D. Detailed view of a blood vessel containing polymorphonuclear cells. E. Secretory cells in the epithelium, staining blue, opaque, or red. F. Intersection between epithelial and dermal tissues, showing polymorphonuclear cells on both sides of the basement membrane. An arrow indicates a polymorphonuclear cell translocating from the dermal to the epidermal compartment, through the basement membrane. G. Control sample of Atlantic Salmon skin. In panels A, B, C, and E, note the red staining of the epithelium and basement membrane in proximity to the louse, indicating fibrin deposition. The basement membrane regains a yellow color (collagen staining) and the epithelium shifts to a lighter shade of blue as the distance from the louse increases. All slides were stained with MOVAT pentachrome, which distinguishes collagen (yellow), glycans (blue), fibrin (bright red), and muscle (red). Abbreviations: Blood vessel (bv), basement membrane (bm), degeneration (deg), dense connective tissue (dct), epithelium (epi), fin ray (ray), scale (sc), vacuolization (vac), polymorphonuclear cells (pmn).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/ec5e05ce46d36ae5c5f8d672.jpg"},{"id":65105610,"identity":"319b2254-f6ab-41de-be0e-cc08cd5b776d","added_by":"auto","created_at":"2024-09-23 16:20:11","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":665978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIHC with Proliferative Cell Nuclear Antigen (PCNA) at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e attachment site in Coho Salmon. \u003c/strong\u003eA. \u0026amp; B. Comparable sections to those shown in Figure 9 (A and B). Panel A shows a tissue section immediately anterior to the \u003cem\u003eL. salmonis\u003c/em\u003e attachment site, while panel B shows a section through the head of \u003cem\u003eL. salmonis\u003c/em\u003e. Despite the relatively thick epithelial tissue at the attachment site, only a few cells were PCNA-positive (PCNA+). These cells were primarily basal cells with a cuboidal shape, located near the basement membrane. C. Epithelial hyperplasia observed at the lice attachment site in one sample (36 hours post-infestation, hpi). Significant positive staining was seen in epithelial cells both at the site directly in contact with the louse and on the opposite side of the fin. Nuclear red staining indicates PCNA+ cells.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/a6acb3448c3ff99f22f4ba59.jpg"},{"id":65106215,"identity":"1ff8a7e9-5fb9-4d3a-b3b7-3e27c762e4c9","added_by":"auto","created_at":"2024-09-23 16:28:11","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":336264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial transcriptomics at \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eattachment site in Coho Salmon at 12 hours post-infestation, reads mapped to the Coho Salmon genome.\u003c/strong\u003e A. UMI counts, reads mapped to the genome of Coho Salmon. B. Differentially expressed genes most specific to the attachment site. Color shows log normalized average expression ratio of the genes listed in the plot. C. Differentially expressed genes at attachment site, as listed in table 3, color shows log normalized expression. Gene name and scale of expression indicated in the plot.\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/0d1978da2153376b1818e54c.jpg"},{"id":65105617,"identity":"2cbd52fe-621b-4efb-ba2d-095d6cb53974","added_by":"auto","created_at":"2024-09-23 16:20:11","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":251044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial transcriptomics reads mapped to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. salmonis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome. \u003c/strong\u003eA. UMI counts mapped to the \u003cem\u003eL. salmonis\u003c/em\u003e genome. B. t-SNE projection of spots, colored by UMI counts. C. K-means clustering into five clusters: Cluster 1 overlapped with Coho Salmon tissue, while Clusters 2 to 5 correspond to spots covered by \u003cem\u003eL. salmonis\u003c/em\u003e. D. t-SNE projection of spots, colored by cluster assignment.\u003c/p\u003e","description":"","filename":"Figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/459efc3ab0fb44d6ebf2b349.jpg"},{"id":65106211,"identity":"a7121b02-d1ab-44b0-aa6b-13e04d484028","added_by":"auto","created_at":"2024-09-23 16:28:11","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":560596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanisms of louse rejection in Coho Salmon.\u003c/strong\u003e Acute inflammation drives the rejection of copepodids. Lice counts on Coho Salmon fins are illustrated (line), while fish with lice id represented by red circles and rejection indicated by horizontal arrows. By 168 hours post-infestation (hpi), visible epithelial hyperplasia in response \u003cem\u003eto L. salmonis\u003c/em\u003e was observed. Experiments sampling tissue with louse beyond 48 hours post-infestation likely samples fish with lower resistance.\u003c/p\u003e","description":"","filename":"Figure13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/600ba6729ca531a82815fe7b.jpg"},{"id":84243195,"identity":"4d0cb73f-40ef-432e-8362-2f27ab3cf96f","added_by":"auto","created_at":"2025-06-09 16:12:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19272128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/01d3dc1b-79a6-4a84-9885-5fac801dd17a.pdf"},{"id":65105608,"identity":"3bfd5fa3-c294-47e9-b21c-ae86201d886d","added_by":"auto","created_at":"2024-09-23 16:20:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":57582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/63726a9fa242355f1f65406d.xlsx"},{"id":65105624,"identity":"82435200-495f-41a2-bcf3-6ea55e4d984c","added_by":"auto","created_at":"2024-09-23 16:20:12","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6622098,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2Frame1Coho.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/dfd32d69a600912460849232.jpg"},{"id":65106212,"identity":"eec1a38e-e16f-4e56-8cbb-5e2149ff5ec8","added_by":"auto","created_at":"2024-09-23 16:28:11","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7733198,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile3Frame2Coho.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/e53a2315a1b13a29701bcc7b.jpg"},{"id":65106216,"identity":"9d522c5c-dfec-4e7c-b698-f2581daafdcf","added_by":"auto","created_at":"2024-09-23 16:28:12","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6795956,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile4Frame3Atlantic.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/5085fd3999688bf5f77e46a7.jpg"},{"id":65106217,"identity":"7af5198e-598d-4b34-8415-1de35195369a","added_by":"auto","created_at":"2024-09-23 16:28:12","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":7599334,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile5Frame4Atlantic.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/c34bd4ed243d488825179e5e.jpg"},{"id":65105626,"identity":"68f3a2ff-39b6-422a-95d2-8dac8d4208ec","added_by":"auto","created_at":"2024-09-23 16:20:13","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":54891796,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile6.tif","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/75475c46715e74ef69aeedcf.tif"},{"id":65105622,"identity":"247392a9-8938-42d3-a80e-500f83ab447f","added_by":"auto","created_at":"2024-09-23 16:20:12","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":13109504,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementartyfile7.tif","url":"https://assets-eu.researchsquare.com/files/rs-4947433/v1/892eda4aa76d393ee9986bb6.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Local inflammation at the salmon louse (Lepeophtheirus salmonis) attachment site drives copepodid rejection in Coho Salmon (Oncorhynchus kisutch)","fulltext":[{"header":"Highlights","content":"\u003cp\u003eSedation aids louse settlement on Salmonid species with more mucous cells.\u003c/p\u003e\n\u003cp\u003eCoho Salmon rejects copepodids\u0026nbsp;after 2 days.\u003c/p\u003e\n\u003cp\u003eSignificant immune response observed in Coho Salmon towards \u003cem\u003eL. salmonis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eInitial louse attachment phase key to resistance.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAtlantic Salmon (\u003cem\u003eSalmo salar\u003c/em\u003e) is one of the most of the most valuable farmed fin fish in aquaculture, but it\u0026rsquo;s sustainable production is hampered by both the salmon louse (\u003cem\u003eLepeophtheirus salmonis\u003c/em\u003e) parasitism and treatments to remove the louse (Fast, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sommerset et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eL. salmonis\u003c/em\u003e exhibits a moderate level of host specificity, primarily infesting fish belonging to the \u003cem\u003eSalmo\u003c/em\u003e and \u003cem\u003eOncorhynchus\u003c/em\u003e genera (Pike \u0026amp; Wadsworth, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Variations in susceptibility to \u003cem\u003eL. salmonis\u003c/em\u003e infestations have been noted between salmonid species (Johnson \u0026amp; Albright, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992a\u003c/span\u003e; Pike \u0026amp; Wadsworth, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) with the pacific species, Coho Salmon (\u003cem\u003eOncorhynchus kisutch\u003c/em\u003e), Pink Salmon (\u003cem\u003eOncorhynchus gorbuscha\u003c/em\u003e) and Chum Salmon (\u003cem\u003eOncorhynchus keta\u003c/em\u003e) regarded as more resistant towards \u003cem\u003eL. salmonis\u003c/em\u003e compared to the highly susceptible Atlantic Salmon (Jones et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sutherland et al., 2014).\u003c/p\u003e \u003cp\u003eThe parasite has a direct life cycle including two free-swimming naupliar stages, one free-swimming infectious copepodid stage, two attached chalimus stages, two preadult stages and an adult stage (Hamre et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Johnson \u0026amp; Albright, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Initially the louse attaches to fin, skin or gills of the fish, and the attachment is facilitated by the free-swimming larval phase. \u003cem\u003eL. salmonis\u003c/em\u003e feeds on fish skin, mucus and blood, and typically, \u003cem\u003eL. salmonis\u003c/em\u003e attachment lesions are characterized by inflammatory changes ranging from ulcerative necrosis, localized hemorrhage and mixed leucocytic infiltration, with comparable responses in the species of \u003cem\u003eOncorhynchus\u003c/em\u003e and \u003cem\u003eSalmo\u003c/em\u003e genera (Fast et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Jones et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In Atlantic salmon, \u003cem\u003eL. salmonis\u003c/em\u003e infestation triggers a rapid and large-scale cellular immune response (Krasnov et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Skugor et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tadiso et al., 2011). The T-cell response is thought to dominate in louse-infested Atlantic Salmon skin (Fast et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Skugor et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), a hypothesis recently supported by single-nuclei RNA sequencing (snRNAseq). Despite this, the acute inflammatory immune response in Atlantic Salmon is ineffective, potentially due to the immunosuppressive nature of \u003cem\u003eL. salmonis\u003c/em\u003e, and rejection does not occur (Braden et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tully \u0026amp; Nolan, 2002). Although various molecules produced by \u003cem\u003eL. salmonis\u003c/em\u003e have been shown to possess immunomodulatory properties (Fast et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Midtb\u0026oslash; et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; \u0026Oslash;verg\u0026aring;rd et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), these analyses have primarily provided targeted data regarding the host-parasite interaction. Moreover, the localized transcriptomic response at the specific point of louse attachment, including differences between epidermal and dermal tissues (Sveen et al., 2021a), is distinct from the broader immune response to the parasite (\u0026Oslash;verg\u0026aring;rd et al., 2023). New methods such as spatial transcriptomics, which covers the transcriptome at spatial resolution, offers a platform to unravel the more complex picture of louse-host pathobiology (Robinson et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). With protocols available for fish skin (Sveen et al., 2023), this approach represents valuable tool for understanding the underlying mechanisms of hos parasite interactions.\u003c/p\u003e \u003cp\u003eUnlike Atlantic salmon, Coho Salmon reject \u003cem\u003eL. salmonis\u003c/em\u003e during initial phases of infestation, believed to result from non-specific host responses, including dermal inflammation as early as 1 day post-infestation (Johnson \u0026amp; Albright, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1992b\u003c/span\u003e), chitin sensing, and immune signaling (Braden et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, cortisol, known for its immune-suppressive effects, increases the susceptibility of Coho Salmon to \u003cem\u003eL. salmonis\u003c/em\u003e infestation when cortisol implants are used (Johnson \u0026amp; Albright, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992a\u003c/span\u003e). Our recent snRNA-seq study further suggests that macrophages may play a key role in the Coho Salmon\u0026rsquo;s immune response towards \u003cem\u003eL. salmonis\u003c/em\u003e (Salisbury et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, Braden et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported elevated expression of \u003cem\u003eclec4e\u003c/em\u003e in Coho Salmon 4\u0026ndash;7 days post-infestation with \u003cem\u003eL. salmonis\u003c/em\u003e. Since \u003cem\u003eclec4e\u003c/em\u003e is a marker for neutrophils in Coho Salmon (Salisbury et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), this finding indicates an increase in neutrophil infiltration at the louse attachment sites. Another proposed mechanism for parasite resistance in Coho Salmon involves hyperplasia of epithelial cells, specifically keratinocytes, along with encapsulation (Braden et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Johnson \u0026amp; Albright, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992a\u003c/span\u003e). However, findings on parasite resistance in Coho Salmon remain somewhat inconclusive, and overall, a similar immune response appears to be activated in both Atlantic Salmon and Coho Salmon skin and fin during lice infestation (Salisbury et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of this study was to identify early resistance mechanisms against \u003cem\u003eL. salmonis\u003c/em\u003e in four salmonid species: Pink Salmon, Coho Salmon, Chum Salmon, and Atlantic Salmon. Samples were collected at four time points within the first 2.5 days post-infestation (dpi): 12-, 24-, 36-, and 48-hours post-infestation (hpi). Due to low attachment success in Pink and Chum Salmon, our focus was directed towards the louse attachment sites on the fins of Atlantic and Coho Salmon. Using a combination of histological techniques, including spatial transcriptomics, we show that the first 48 hpi are critical for copepodid rejection in Coho Salmon, with \u003cem\u003eL. salmonis\u003c/em\u003e being expelled through an acute inflammatory response. These findings highlight the species-specific immune mechanisms underlying early host resistance to parasite infestation.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e1.1 Source of fish\u003c/p\u003e \u003cp\u003eCoho Salmon (1\u0026ndash;2 g) and Pink Salmon (0.25\u0026ndash;0.3 g) were transferred from Quinsam River Hatchery, Nanaimo, British Columbia, and Chum Salmon were transferred at ~\u0026thinsp;0.6 g from Inch Creek Hatchery, Dewdney, British Columbia, to the same Level III Quarantine Facility (CATC), Prince Edward Island, Canada (March 2021). This involved packaging in chilled coolers containing freshwater from the source, supersaturated with oxygen (ca. 250%), and a transport density of 75\u0026ndash;100 g / (30 X 15 X 16-inch shipping boxes), overnight (\u0026lt;\u0026thinsp;36 hr transit time). Two thousand Atlantic Salmon eggs from 10 families were donated to the project by StofnFiskur HF (Benchmark, Iceland) and synchronized to 360 \u0026deg;d at delivery and shipped chilled on ice to The Center for Aquaculture Technologies Souris Canada (CATC) in late November 2020.\u003c/p\u003e \u003cp\u003e1.2 Animal husbandry\u003c/p\u003e \u003cp\u003eFish were acclimated to ambient recirculating conditions (freshwater, 10\u0026ndash;12\u0026deg;C) over several hours in 150 L tanks. All four species were maintained under these conditions (12 h light: 12 h dark, 2\u0026ndash;4 L / min flow, 90\u0026ndash;110% DO, and fed 2\u0026ndash;3 times per day to satiation) until they reached the target weight for the lice challenge test experiment (ca. 25 g). When the population average weight reached 15 g, along with visual indicators of smoltification such as loss of parr marks, Instant Ocean\u0026reg; (Instant Ocean, Blacksburg, USA) was slowly added to the partially recirculating system such that salinity was increased by 4\u0026ndash;5 ppt per day to achieve 32\u0026ndash;33 ppt in 7 days. Fish were fed to satiation 2\u0026ndash;3 times daily with a commercially available diet (Skretting; Skretting, #2 Crum initially and later 2.3 Nutra RC). In total 600 na\u0026iuml;ve fish, 150 of each salmonid species: Atlantic, Coho, Pink and Chum Salmon, were incorporated into a lice challenge trial at CATC, Canada. On the day of distribution to the study tanks, bulk weights of a minimum of 50 individuals per species were recorded (Average size: Atlantic salmon 40.0 g; Coho 12.5 g, Pink 50.1 g, Chum 80.6 g). All fish were in good health prior to being enrolled in the study.\u003c/p\u003e \u003cp\u003eThe experimental setup involved tanks that were supplied with recirculating aquaculture system (RAS) saltwater at a concentration of 32\u0026ndash;37 ppt. The holding temperature was maintained at 12\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C throughout the study. A total of 20 tanks with a combined capacity of 135 L were used. Fish of each of the salmonid species were distributed over 5 tanks (3 tanks with infestation and 2 control tanks), giving 30 fish per replicate tank and 150 fish in total per species. For the experimental tanks, the flow rate of the water within the tanks was maintained at around 2\u0026ndash;4 liters per minute, ensuring approximately one tank exchange per hour. Dissolved oxygen saturation levels were maintained between 90\u0026ndash;120% to ensure adequate oxygenation for the fish. A standard photoperiod of 12 hours of light followed by 12 hours of darkness was implemented to simulate natural light conditions. This light cycle was regulated throughout the study. Fish were fed to satiation once daily with commercial pelleted diet appropriate for the fish size throughout the study period. Salinity, temperature, and dissolved oxygen were maintained within specified ranges. Fish were monitored for mortality and / or signs of morbidity twice daily as a standard husbandry procedure. Before the final challenge (challenge 3), fish were transferred from the RAS to a flow through system. However, tank size, water flow and light intensity remained identical to that of the RAS.\u003c/p\u003e \u003cp\u003e1.3 Origin and cultivation of \u003cem\u003eL. salmonis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eEgg strings from adult female lice (\u003cem\u003eL. salmonis\u003c/em\u003e) were collected from Atlantic salmon farms located in the Bay of Fundy, Atlantic Canada. Copepodids were generated by incubating the strings in a static laboratory seawater system. Concentration and viability of copepodids were validated under a stereo light microscope immediately before each challenge. The copepodids were aliquoted for challenge exposure in each individual tank. The challenge duration and concentration of copepodids per fish was adjusted depending on the challenge test methodology (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eOverview of the challenge experiments\u003c/b\u003e. Fish species, number of fish, challenge method, exposure duration and \u003cem\u003eL. salmonis\u003c/em\u003e concentration during each challenge.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFish species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e#Fish challenged\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e# Tanks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChallenge method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChallenge duration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eL. salmonis\u003c/em\u003e concentration (#/fish)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eChallenge 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e13th of Oct\u003c/p\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAtlantic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eStatic in 100L container *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e3 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e82.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eChallenge 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e16th of Oct 2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAtlantic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eStatic in tanks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e2 hours ˟\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e149.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eChallenge 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e26th of Oct 2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAtlantic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eStatic in 10L recovery bath \u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e15 minutes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e49.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePink\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c10\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eFish were equally distributed into challenge tanks that served as replicates. Additional control tanks without any lice exposure were utilized for every species at each challenge.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e* Fish were netted out and challenged outside the tank in a 100L container. After challenge, fish were netted back to its respective tank.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c10\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003e\u0026Dagger;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eFish were netted out, anesthetized, and recovered in 10L bath with lice. Post challenge entire content of a bucket including fish, lice, and water were added back to respective tank.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e˟ Water supply resumed after 3 hours of static challenge.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e1.4 Challenge experiments\u003c/p\u003e \u003cp\u003eIn total three consecutive immersion challenge trials on the same batch of fish were performed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and lice production and challenge methodologies used a combination of those described by (Jones et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Poley et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Briefly, in the first challenge, 12 tanks (30 fish / tank) were infested with \u003cem\u003eL. salmonis\u003c/em\u003e copepodids. Prior to exposure, the fish were netted from their original tanks and equally distributed into 100 L challenge containers that served as replicates. The fish were challenged with 82.2 copepodids / fish in static 100 L containers for 3 h. Additional control tanks without any lice exposure were utilized for every species at each challenge. Supplemental oxygen was added during the infestation procedure to maintain 6.0\u0026ndash;9.0 mg / L O\u003csub\u003e2\u003c/sub\u003e for the 3 h exposure period. After the 3 h exposure, fish were transferred to their original fish tanks. Since the first challenge did not produce sufficient infestation levels in Chum Salmon Chum and Pink Salmon, a second challenge was carried out. This challenge was similar to the first challenge, but with reduced fish number (25 fish / tank), due to sampling a subset of fish each time to examine challenge success and a higher lice concentration, 149.3 lice / fish for 2 h. Again, a low number of lice were observed in Chum and Pink Salmon. In both the first and second challenge, Pink and Chum Salmon exhibited hyperactive swimming activity. Due to low numbers of lice in the first two challenges the exposure method was modified for the 3rd challenge. During the 3rd challenge each tank of fish was anesthetized in TMS (100 mg / L) and the anesthetized fish recovered in a static system containing lice. In total, 22 fish / tank were netted out, anesthetized, and recovered in 10 L baths with \u003cem\u003eL. salmonis\u003c/em\u003e at a concentration 49.8 lice / fish for 15 min. Dissolved oxygen was supplemented using air-stones during the lice challenge tests. Post challenge the entire content of the 10 L container, including fish, lice, and water were added back to the respective tank. To maintain an adequate number of challenged fish, in addition to the original lice exposure tanks, one control tank was utilized in Challenge 3. For each challenge, control tanks contained the same number of fish as the lice exposure tanks for each species. Control fish were handled the same as the challenged fish in every possible way except being exposed to the lice.\u003c/p\u003e \u003cp\u003e1.5 Lice counting and tissue sampling\u003c/p\u003e \u003cp\u003eTissue sampling and lice counts were performed at six time points post infestation, 12 hpi, 24 hpi, 36 hpi, 48 hpi, 60 hpi and 168 hpi, while na\u0026iuml;ve control samples were collected at 36 and 168 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For tissue sampling, fish (n\u0026thinsp;=\u0026thinsp;3 / tank; n\u0026thinsp;=\u0026thinsp;9 per species per time point) were mildly sedated using an anesthetic dose (100 mg / L) of TMS (Syndel, Nanaimo, BC, Canada) and then were administered a lethal blow to the head. Individual fish were placed in a separate container so that lice were not lost during processing. Fish were processed prior to lice counts by bleeding, and each opercula, gill arch, and fin was placed in a petri dish containing sea water. Finally, attached \u003cem\u003eL. salmonis\u003c/em\u003e were counted by life stage and tissue type (body, fins, or gills) using a stereomicroscope. After the final challenge, the study continued until 168 hpi, at which point all remaining fish (n\u0026thinsp;=\u0026thinsp;7 per tank) were sampled as above. Skin was collected from a standardized position on the flank of the fish (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and transferred to 10% buffered formalin (Fisher-Scientific, Missassauga, ON, Canada) for histological analysis. Fins with lice attached were transferred to 10% buffered formalin (Fisher-Scientific, Missassauga, ON, Canada), or embedded in O.C.T Embedding Matrix (CellPath, UK) as described by Sveen et al 2023. A full list of lice counts, and samples collected during the challenge experiments, including those for genetic analysis, may be found in Supplementary file 1. Samples for histological analysis and spatial transcriptomics were shipped from Canada to Norway using Cencora World Currier\u0026trade; with temperature loggers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e1.6 Processing of tissue samples for histological examination\u003c/p\u003e \u003cp\u003eEmbedding, sectioning, and staining of the tissue samples (skin and fin) was done at the Veterinary Institute in Harstad, Norway. For the fin samples with \u003cem\u003eL. salmonis\u003c/em\u003e attached the samples were trimmed under a stereoscope at the position of the louse (2 x 3 mm) prior to embedding the samples. Tissue samples with registered louse at sampling, but no visual observation of the louse during examination in the laboratory were excluded from the analysis, resulting in a total of 15 samples with attached louse in Atlantic Salmon and Coho Salmon (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Paraffin embedded tissue sections were hydrated in water and stained with 1% Alcian blue (AB, Alfa Aesar) in 3% acetic acid for 15 min, transferred to 1% periodic acid (VWR) for 10 min, followed by Schiffs (Sigma-Aldrich\u0026reg;, Saint-Louis, MO, USA) reagent for 15 min, and finally for 30 s in hematoxylin (VWR, Radnor, PA, USA) before dehydration, mounting and AB / PAS (Alcian Blue / Periodic acid \u0026ndash; Schiffs) staining (which stains mucous cells blue, purple or pink based on the acidity of the mucins). MOVAT staining was carried out on one sample (Coho Salmon) with lice attached, flowing the protocol outlined by (Suvarna et al., 2018). The stained tissue sections were scanned at 20 X with Hammamatsu Nanozoomer S360 and uploaded in the Aiforia\u0026reg; software for visualization. Selected sections were viewed and photographed at higher magnifications (40 X, 60 X and 100 X) with Zeiss Axio Observer Z1 (Zeiss, Oberkochen, Germany). The sections were analyzed by a trained histologist. Multiple parallel sections were done for each sample, leaving no extra sample material for additional analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eFin samples with\u003c/b\u003e \u003cb\u003eL. salmonis\u003c/b\u003e \u003cb\u003eattached processed for histological analysis.\u003c/b\u003e The sampled lice attachment site varied, and is denoted as body flank (BF), caudal fin (C), pectoral fin (PC), adipose fin (A) or dorsal fin (D)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"17\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"17\" nameend=\"c17\" namest=\"c1\"\u003e \u003cp\u003eAtlantic salmon\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ehpi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cb\u003e168\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u003cb\u003e168\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u003cb\u003e168\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eBF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"17\" nameend=\"c17\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCoho Salmon\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ehpi\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u003cb\u003e48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u003cb\u003e168\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e1.7 Immunohistochemistry\u003c/p\u003e \u003cp\u003eImmunohistochemistry (IHC) with primary antibodies for proliferating cell nuclear antigen (PCNA) and Tumor Necrosis Factor Alpha (TNF-α) (Vertebrate Antibodies, UK) was carried out on a total of six selected samples from Coho Salmon, three samples for each primary antibody. In brief, immunohistochemistry (IHC) was performed by blocking de-paraffinized tissue sections with horse serum (Sigma-Aldrich, Missouri, USA) for 20 min, followed by primary antibody, being either proliferation nuclear cell antigen (dilution 1 : 60), or TNF-α (1 : 2000). Slides were washed twice in PBS before incubating with ImmPress AP-anti-Rabbit IgG for TNF-α (Vector Laboratories) or AP-anti-mouse IgG for TNF-α (Vector Laboratories) for 30 min. Immpact Vector Red substrate kit (Vector Laboratories) with alkaline phosphatase was used for 30 min according to the protocol before counterstaining 30 s with haematoxylin (Sigma). Slides were visualized as described in the previous section.\u003c/p\u003e \u003cp\u003e1.8 AI \u0026ndash; analysis of skin from body flank\u003c/p\u003e \u003cp\u003eSkin samples from the body flank were analyzed using Nofima\u0026rsquo;s skin-AI model on the Aiforia\u0026reg; platform, as outlined by (Sveen et al., 2021b). The analysis included tissue samples from the following four species and time points: Atlantic Salmon (N\u0026thinsp;=\u0026thinsp;6 control, N\u0026thinsp;=\u0026thinsp;9 infested (4 samples 12 hpi, 5 samples 36 hpi)), Chum Salmon (N\u0026thinsp;=\u0026thinsp;10 control, N\u0026thinsp;=\u0026thinsp;14 infested (8 samples 12 hpi and 6 samples 36 hpi)), Coho Salmon (N\u0026thinsp;=\u0026thinsp;9 control, N\u0026thinsp;=\u0026thinsp;13 infested (6 samples 12 hpi, 7 samples 36 hpi)), Pink Salmon (N\u0026thinsp;=\u0026thinsp;10 control, N\u0026thinsp;=\u0026thinsp;15 infested (7 samples 12 hpi, 8 samples 36 hpi)). Post-AI-analysis, the results were exported, filtered as described by Sveen et al. 2021b, and further analyzed in R. Given no significant differences in skin parameters between the infestation at the two timepoints (12 hpi and 36 hpi), these were combined for the final analysis, comparing infestation and species. Statistical calculations included two-way ANOVAs, Tukey post-hoc tests, and Pearson correlations, using the provided stats package functions (aov(), TukeyHSD(), and cor.test()). The results were visualized using a combination of custom functions and the beeswarm package.\u003c/p\u003e \u003cp\u003e1.9 Spatial transcriptomic of lice attachment site in Coho Salmon\u003c/p\u003e \u003cp\u003eIn total four samples, two Coho Salmon fins, and two Atlantic salmon fins with \u003cem\u003eL. salmonis\u003c/em\u003e attached were processed for spatial transcriptomics, as described by (Sveen et al., 2023). Libraries were sequenced on NovaSeq 6000 SP flow cell (Illumina, USA) at the Norwegian Sequencing Center as 50 bp paired end reads. Sequencing was done using the following cycles: read 1, 28 cycles, i7 Index; 10 cycles, i5 Index; 10 cycles; and read 2, 90 cycles (Visium Spatial Gene Expression User Guide; 10x Genomics). Reads were aligned to the Atlantic Salmon genome (version Ssal_v3.1, INSDC Assembly GCA_905237065.2), Coho Salmon Genome build Okis_V2, accession number GCA_002021735.2 and \u003cem\u003eL. salmonis\u003c/em\u003e Genome build UVic_Lsal_1.0, accession number GCA_016086655.3 () using the software Space Ranger (version 1.3.1; 10x Genomics, USA). Sequence data have been submitted to NCBI's Sequence Read Archive (SRA) as BioProject PRJNA1149653. High-resolution JPG images (Supplementary file 2\u0026ndash;5) from each of the associated tissue sections were aligned to the reads by default settings. Optimal number of tissue clusters and cluster membership of spots was defined using graph-based clustering [modified python implementation of the augmented implicitly restarted Lanczos bidiagonalization algorithm (IRLBA) (Baglama and Reichel 2005)] in Space Ranger. Transcripts defining tissue clusters were filtered using the following criteria: only upregulated transcripts (relative to the other clusters), adjusted P-value (Benjamini\u0026ndash;Hochberg procedure)\u0026thinsp;\u0026lt;\u0026thinsp;0.1, and mean barcoded unique molecular identifier (UMI) count\u0026thinsp;\u0026gt;\u0026thinsp;1. Expression levels of genes were visualized using 10x Genomics Loup Browser v8.0.0. Fine tuning of clustering was done using k-means in 10x Genomics Loup Browser v8.0.0. Searching for differentially expressed genes at the host parasite interphase using k-mean function. For \u003cem\u003eL. salmonis\u003c/em\u003e K-mean were set to 5 and differentially expression ran excluding cluster 1. Further, top 100 DEG for cluster 3, 4 and 5 (host / parasite interface), were exported followed by manual curation for genes involved in immune function / regulation.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e2.1 \u003cem\u003eL. salmonis\u003c/em\u003e count\u003c/p\u003e\n\u003cp\u003eAfter the first two challenge exposures there were no copepodids found on any Pink or Chum Salmon and only a limited number of copepodids found on Atlantic salmon (mean\u0026thinsp;=\u0026thinsp;8.0 +/- 6.4 SD) and Coho Salmon (mean\u0026thinsp;=\u0026thinsp;1.0 +/- 1.0 SD). These infestations did not progress past 8 days. In the third challenge, the total average lice count on Atlantic Salmon was 13.44 / fish (SD 4.52) at 12 hpi, while total average counts in Coho, Pink, and Chum Salmon were 3.89 / fish (SD 1.79), 2.89 / fish (SD 4.31), and 5 / fish (SD 2.05) respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Total counts in Pink and Chum Salmon decreased to 0.44 (SD 0.96) and 1.40 (SD 1.08) respectively at 24 hpi, and both species dropped to 0 lice / fish by day 7 (termination). Most of the settled lice on Atlantic Salmon progressively moulted to later developmental stages, e.g. chalimus at 168 hpi. At termination, the total average lice count on Coho and Atlantic Salmon were 0.62 and 11.24 lice / fish respectively. Notably, lice count on Coho fins was initially higher than on any of the other salmonid species but was only sustained for 48 hpi before decreasing to 0.57 lice on fins per fish at termination.\u003c/p\u003e\n\u003cp\u003e2.2 Quantitative assessment of fish skin\u003c/p\u003e\n\u003cp\u003eQuantitative histological analysis of skin from the body flank of the fish showed no significant differences in the epithelial tissue or mucous cell number between infested and control samples within each salmonid species (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). However, there were differences between the species. Chum Salmon had the largest area of epithelial tissue per mm of skin, and 30\u0026ndash;40% of the epithelial area was mucous cells, corresponding to more than 100 mucous cells per mm of skin. Additionally, mucous cells accounted for 15\u0026ndash;20% of the epithelial surface in Pink and Coho Salmon, while Atlantic Salmon showed the lowest proportion, with only 10% coverage.\u003c/p\u003e\n\u003cp\u003eThere is ongoing debate in the literature regarding which salmonid species is most resistant to salmon lice infestation. Experimental trials with \u003cem\u003eL. salmonis\u003c/em\u003e have shown that Chum Salmon are more susceptible to lice compared to Pink Salmon (Jones et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Atlantic Salmon (Sutherland et al., 2014). However, another study found that wild Pink Salmon exhibited a higher lice prevalence compared to Chum Salmon (Nagasawa, \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). Notably, under experimental conditions, both Chum Salmon and Pink Salmon must be sedated to facilitate infestation. Hence, based on our findings, we suggest that mucous cells, potentially in combination with behavioral mechanisms (disrupted by sedation), may offer protection against lice settlement in Chum and Pink Salmon. Additionally, factors beyond mucous cell count, such as variations in presence of immunological defense molecules (Esteban, \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e), or organic compounds (Difford et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) in the fish mucus, could contribute to \u003cem\u003eL. salmonis\u003c/em\u003e resistance and warrant further investigation.\u003c/p\u003e\n\u003cp\u003e2.3 Assessment of the attachment site of \u003cem\u003eL. salmonis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGiven that \u003cem\u003eL. salmonis\u003c/em\u003e successfully attached to the fins of both Atlantic Salmon and Coho Salmon, but only Coho Salmon displayed copepodid rejection, we directed our investigation towards a more detailed analysis of the attachment sites in these two species. In Atlantic Salmon signs of tissue damage inflicted by lice were apparent as early as 12 hpi, with epithelial tissue erosion and a reduction in mucous cells directly beneath the mouth tube of \u003cem\u003eL. salmonis\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Concurrently, an influx of polymorphonuclear cells was observed within dermal blood vessels beneath the attachment site. Over later time intervals (36\u0026ndash;168 hpi), there were no polymorphonuclear cells directly at the feeding site. This suggests that the inflammatory response at later stages may have been reduced by the louse\u0026rsquo;s immunomodulatory effects (Braden et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hamilton et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Midtb\u0026oslash; et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; \u0026Oslash;verg\u0026aring;rd et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Further, lice induced tissue damage included the depletion of mucous cells beneath the louse and erosion of the epithelium (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), aligning with previous research (Tully \u0026amp; Nolan, 2002). Although most samples showed a decrease in mucous cell abundance at the feeding site, some exceptions were noted. Notably, two Atlantic Salmon samples had a significant increase in purple-staining mucous cells at the louse attachment site, with similar observations in Coho Salmon (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplementary file 1). In healthy Atlantic salmon skin, purple mucous cells are typically sparsely distributed (Sveen et al., 2021b), but lice infestation may increase in their prevalence at attachment site (Sveen et al., 2021a). The variation in mucous cell color holds significance for mucus function, indicating a glycosylation shift in mucin proteins (Zaretsky \u0026amp; Wreschner, 2013). Transitioning from blue-staining to purple-staining mucous cells, with Alcian blue and PAS staining, suggests alterations in glycans on the mucin backbone, potentially resulting in changes to mucus pH, solubility, and its capacity to interact with and bind to pathogens, such as bacteria (Padra et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Padra et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Padra et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, several pathogens, both bacteria and parasites, can employ a strategy known as \u0026quot;glycan gimmickry,\u0026quot; wherein they produce glycans that mimic those of the host. This mimicry can facilitate interaction with host lectins, potentially modulating or evading immune responses (Rodrigues et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, the dynamics of the mucous cell population and mucin glycan pattern could shed light on mechanisms underlying host defense evasion and potentially inform novel intervention strategies.\u003c/p\u003e\n\u003cp\u003eCoho Salmon demonstrated a heightened immune response towards \u003cem\u003eL. salmonis\u003c/em\u003e, particularly between 36 and 48 hpi, which corresponded to the time point before the observed reduction in lice numbers. Among the ten samples analyzed in this time interval, four exhibited a robust influx of polymorphonuclear cells not only within blood vessels but also infiltrating the dermal tissue (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Remarkably, in three of these samples, polymorphonuclear cells filled the entire mesenchymal cavity of the fin contrasting sharply with the inflammatory reaction observed in Atlantic Salmon. Neutrophils were initially identified as the predominant cell type at the site of louse attachment in both Atlantic Salmon and Coho Salmon through histological techniques (Johnson \u0026amp; Albright, \u003cspan class=\"CitationRef\"\u003e1992a\u003c/span\u003e), however precise classification of immune cells by visual examination can be difficult. Analysis of raw and normalized snRNA-seq counts for inflammatory cells at the attachment site in fin and skin samples (Supplementary file 6; data from Salisbury et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) show an increased proportion of both neutrophils and macrophages at 12 hpi in Coho Salmon fin samples. This increase was not observed in Coho Salmon skin, Atlantic Salmon fin, or Atlantic Salmon skin samples. Additionally, samples with significant infiltration of polymorphonuclear cells also showed positive staining for anti-TNF\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e), a cytokine predominantly produced and released by activated macrophages that plays a central role in recruiting leukocytes to inflamed tissues.\u003c/p\u003e\n\u003cp\u003eIn one sample, the cephalon (head) of the louse was precisely captured at the attachment site, offering insights into how Coho Salmon may reject or combat \u003cem\u003eL. salmonis\u003c/em\u003e infestations at the copepodid stage (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Notably, polymorphonuclear cells were observed in the epithelial compartment near the \u003cem\u003eL. salmonis\u003c/em\u003e head and were abundant in the dermal compartments directly beneath the louse. The characteristics of these cells varied depending on their location; those closest to the louse exhibited fragmented nuclei within a single cell body (Supplementary file 7), which may indicate they are undergoing apoptosis, a common feature of activated immune cells (Donovan et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ekert \u0026amp; Vaux, \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e). Moreover, significant alterations in the epithelial tissue and basement membrane were observed at the louse attachment site, highlighted by red staining with MOVAT pentachrome (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). In contrast, both the basement membrane and epithelium retained normal staining at increasing distances from the \u003cem\u003eL. salmonis\u003c/em\u003e head. This red staining may indicate fibrinoid necrosis (Movat, \u003cspan class=\"CitationRef\"\u003e1960\u003c/span\u003e), a specific type of tissue necrosis characterized by the deposition of fibrin-like proteinaceous material, commonly associated with immune complex-mediated diseases and inflammatory responses (Bajema \u0026amp; Bruijn, \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e; Movat \u0026amp; More, \u003cspan class=\"CitationRef\"\u003e1957\u003c/span\u003e). Previous studies on louse-infested Coho Salmon have also reported necrotic tissue at louse attachment sites (Johnson \u0026amp; Albright, \u003cspan class=\"CitationRef\"\u003e1992b\u003c/span\u003e). Additionally, this region displayed signs of hyalinization, edema and epithelial vacuolization, indicating that the strong inflammatory response leads to tissue damage. The extent of this damage suggests that active degranulation may be occurring (Hirsch \u0026amp; Cohn, \u003cspan class=\"CitationRef\"\u003e1960\u003c/span\u003e). Degranulation typically involves the release of pro-inflammatory factors, such as histamines, leukotrienes, and cytokines (Gierlikowska et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), which target invading pathogens but can also cause collateral damage to host tissues. Following degranulation, immune cells often undergo apoptosis to limit excessive tissue damage and inflammation (Soehnlein et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). An apoptotic process will affect the cell nuclei, and may partly explain why granulocytes were difficult to detect in our snRNAseq analysis (Salisbury et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFinally, immunohistochemistry with PCNA (a marker for cell proliferation) was performed on two samples of Coho Salmon: one featuring the captured head of the louse, and the other exhibiting notable epithelial tissue hyperplasia towards \u003cem\u003eL. salmonis\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). PCNA\u0026thinsp;+\u0026thinsp;cells were predominantly observed in the sample displaying hyperplasia, but not in the sample with abundance of polymorph nuclear cells. Localized hyperplasia of the epithelial tissue and keratinocyte proliferation, occasionally leading to the encapsulation of entire lice, has been generally recognized as a pivotal factor driving lice resistance in Coho Salmon (Johnson \u0026amp; Albright, \u003cspan class=\"CitationRef\"\u003e1992a\u003c/span\u003e; Salisbury et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, within the timeframe of 36 to 48 hours post-infestation in the present study, only one sample exhibited this characteristic. This may suggest that it is the acute inflammatory response, and not epithelial hyperplasia, is the primary mechanism acting against copepodids in Coho Salmon.\u003c/p\u003e\n\u003cp\u003e2.4 Transcriptomic responses at the intersection of louse and host\u003c/p\u003e\n\u003cp\u003eTo further characterize the host responses to \u003cem\u003eL. salmonis\u003c/em\u003e, we employed spatial transcriptomics on Atlantic and Coho Salmon fins with \u003cem\u003eL. salmonis\u003c/em\u003e attached in the first hours after infestation. We were only able to detect tissue from \u003cem\u003eL. salmonis\u003c/em\u003e and salmon tissue on one slide, mainly due to the technical difficulty of pinpointing the exact point of attachment between the host tissue and the small \u003cem\u003eL. salmonis\u003c/em\u003e copepodid which is less than 1 mm in size at the copepodid stage. Further, given that differentially expressed genes are determined within each tissue slide rather than across multiple samples, we directed our analysis towards the specimen containing both louse and host tissue and manually curated for annotated genes that were DEGs at the intersection between the host and the parasite. This analysis was performed separately for the Coho Salmon genome and the \u003cem\u003eL. salmonis\u003c/em\u003e genome.\u003c/p\u003e\n\u003cp\u003e2.4.1 Spatial transcriptomic slide mapped to the genome of Coho Salmon\u003c/p\u003e\n\u003cp\u003eTissue (\u003cem\u003eL. salmonis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Coho Salmon) covered 250 bar coded spots. For the Coho Salmon total genes detected were 18218 and the median genes per spot were 1252. The UMI counts overlapped with both the Coho Salmon tissue and \u003cem\u003eL. salmonis\u003c/em\u003e tissue, however with lowest count on the \u003cem\u003eL. salmonis\u003c/em\u003e tissue (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA). This overlap could potentially be due to the feeding behavior of \u003cem\u003eL. salmonis\u003c/em\u003e on host tissue, including consumption of immune cells at the point of attachment. Manual curation for differentially expressed genes at the attachment site resulted in 17 genes showing high specificity at the host-parasite interface (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), with eight of these being most specific to the area of attachment (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB and C).\u003c/p\u003e\n\u003cp\u003eThe differentially expressed genes at the attachment site were associated with immune responses, oxidative stress responses, proteolysis, and cell migration, all characteristic of acute inflammation (Soliman \u0026amp; Barreda, \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Genes involved in immune responses and proteolysis included \u003cem\u003egranulocyte colony-stimulating factor receptor\u003c/em\u003e (\u003cem\u003ecsf3r\u003c/em\u003e), \u003cem\u003emyeloperoxidase\u003c/em\u003e (\u003cem\u003empx\u003c/em\u003e), \u003cem\u003elysozyme\u003c/em\u003e (\u003cem\u003elyz\u003c/em\u003e), \u003cem\u003ematrix metalloproteinase 9\u003c/em\u003e (\u003cem\u003emmp9\u003c/em\u003e), \u003cem\u003ecathepsins\u003c/em\u003e (\u003cem\u003ectss\u003c/em\u003e), \u003cem\u003emetalloproteinase inhibitor 2-like\u003c/em\u003e (\u003cem\u003etimp2\u003c/em\u003e), and \u003cem\u003earachidonate 5-lipoxygenase\u003c/em\u003e (\u003cem\u003ealox5a\u003c/em\u003e). Notably, \u003cem\u003emyeloperoxidase\u003c/em\u003e, an enzyme critical for the antimicrobial activity of neutrophils and \u003cem\u003egranulocyte colony-stimulating factor receptor\u003c/em\u003e, a receptor that stimulates neutrophil production, were highlighted. Furthermore, inflammatory cells, particularly neutrophils and macrophages, are known to have high concentrations of proteases, including lysozyme, matrix metalloproteinases, and cathepsins (Soliman \u0026amp; Barreda, \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e), hence the upregulation of \u003cem\u003emetalloproteinase inhibitor 2-like\u003c/em\u003e is likely to counterbalance protease activity. Further, the production of reactive oxygen species (ROS) is essential for antimicrobial activity and immune signaling. Genes such as \u003cem\u003eglutathione peroxidase 1b\u003c/em\u003e (\u003cem\u003eGPX1b\u003c/em\u003e) and \u003cem\u003emetallothionein\u003c/em\u003e suggest active antioxidant defenses at the attachment site, providing protection against ROS. Additionally, the expression of \u003cem\u003ethymosin beta 4\u003c/em\u003e and \u003cem\u003ejunB proto-oncogene\u003c/em\u003e, which are involved in various cellular processes including cell migration, angiogenesis, and wound healing (Sveen et al., 2019; Sveen et al., 2018), suggests activation of cell migration. Finally, in our recent single-cell study, \u003cem\u003emmp9\u003c/em\u003e was identified as a marker of neutrophils in Atlantic Salmon, while \u003cem\u003ecsf3r\u003c/em\u003e (ENSOKIG00005018820) was found to be highly expressed in both neutrophils and macrophages (Salisbury et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). A different paralogue of \u003cem\u003ectss\u003c/em\u003e (i.e., ENSOKIG00005022279) than the one detected here is also known as a macrophage marker (Salisbury et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Hence, the upregulation of these genes, therefore, supports the infiltration of macrophages and neutrophils to the attachment site of\u0026nbsp;\u003cem\u003eL. salmonis\u003c/em\u003e in Coho Salmon.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDifferentially expressed genes at \u003cem\u003eL. salmonis\u003c/em\u003e attachment site in Coho Salmon.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSymbol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005043004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ealox5ap\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003earachidonate 5-lipoxygenase-activating protein-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005021726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ectss\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ecathepsin S-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005007964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ecoro1a\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ecoronin-1A-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005049844\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003empx\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eeosinophil peroxidase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005011185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003efbp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003efructose-1,6-bisphosphatase 1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005040066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eGpia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eglucose-6-phosphate isomerase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005014626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003egpx1b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eglutathione peroxidase 1b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005018820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ecsf3r\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003egranulocyte colony-stimulating factor receptor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005018202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ejunb\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eJunB proto-oncogene, AP-1 transcription factor subunit\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005050890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003elyz\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003elysozyme [Source:NCBI gene\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005010179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003emmp9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ematrix metalloproteinase-9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005043531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003emmp9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ematrix metalloproteinase-9-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005030083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003emt2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003emetallothionein B\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005046918\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003enccrp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNCCRP1, F-box associated domain containing\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005018695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eoscp1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eprotein OSCP1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005035017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003etimp2b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003emetalloproteinase inhibitor 2-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSOKIG00005025750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003etxnip\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ethioredoxin-interacting protein-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e2.4.2 Spatial transcriptomic slide mapped to the \u003cem\u003eL. salmonis\u003c/em\u003e genome\u003c/p\u003e\n\u003cp\u003eA total of 8885 genes were detected for \u003cem\u003eL. salmonis\u003c/em\u003e. The louse tissue covered only 12 barcoded spots on the spatial transcriptomics slide, corresponding to the approximate size of the parasite (500 \u0026micro;m). The UMI counts mapped to these 12 spots ranged from 8000 to 34372 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eA and B). Analysis of differentially expressed genes (DEGs) at the host-parasite interface, particularly those related to regulation and immunity, identified 14 louse genes with a fold change greater than 4 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). While some of these genes are directly associated with immune responses (e.g., \u003cem\u003etlr7\u003c/em\u003e, \u003cem\u003enotch1b\u003c/em\u003e), others may influence immune-related pathways indirectly or be involved in broader regulatory processes within the parasite. The upregulation of these genes could indicate adaptations of the parasite to its host environment, including mechanisms to evade host immune responses or modulate host cell functions (\u0026Oslash;verg\u0026aring;rd et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, given that only a single specimen was analyzed and the \u003cem\u003eL. salmonis\u003c/em\u003e transcriptome remains difficult to interpret due to a poorly annotated genome (with over 13.000 protein-coding genes, many of which are uncharacterized), we are cautious in drawing definitive conclusions. Nevertheless, this study demonstrates the feasibility of capturing the transcriptomes of two different species on a single spatial transcriptomics slide. With further refinement, this methodology could provide novel insights into co-regulation and cross-species signaling mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Differentially expressed \u003cem\u003eL. salmonis\u003c/em\u003e genes at the attachment Site.\u0026nbsp;\u003c/strong\u003eCluster 2 represents 12 barcoded spots corresponding to\u0026nbsp;\u003cem\u003eL. salmonis\u003c/em\u003e tissue. Clusters 3, 4, and 5 each consist of a single barcoded spot located at the host-parasite interface (refer to Figure 12). Red shading indicates positive fold changes, while blue indicates negative fold changes relative to other clusters.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e2.5 Concluding remarks\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe observed infiltration of polymorphonuclear cells at attachment sites in Coho Salmon, coupled with the upregulation of genes related to immunity at the direct interface between the host and parasite, indicates a concerted effort by the immune system to combat the invading parasite. However, it\u0026apos;s important to acknowledge that this study is based on a limited number of samples, primarily due to challenges in obtaining high-quality sections from verified louse attachment sites. Despite the relatively small sample size, compelling evidence supports an acute inflammatory response characterized by the recruitment of neutrophils and macrophages, leading to the effective expulsion of copepodids in Coho Salmon.\u003c/p\u003e\n\u003cp\u003eMoreover, Coho Salmon likely employs multiple resistance strategies, combining a strong inflammatory response against copepodids with epithelial hyperplasia, which may become more important when copepodid rejection is incomplete (Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e). When considering the challenge of developing resistance, it\u0026rsquo;s important to recognize that overcoming two different genetic resistance mechanisms requires at least two types of adaptations in the pathogen. A single variant or mutation is insufficient for the louse to survive and reproduce successfully. This means that if Coho Salmon possess multiple resistance mechanisms against the louse, it will take significantly longer and be much more difficult for the louse to develop resistance. This could explain why we haven\u0026rsquo;t observed resistance development in \u003cem\u003eL. salmonis\u003c/em\u003e towards Coho Salmon. This study demonstrates that acute inflammation targeting the copepodid stage is likely the primary mechanism driving salmon lice resistance in Coho Salmon. Moreover, some of the identified genes and pathways at the attachment site in Coho Salmon could be promising candidates for gene editing or modulation to assess their effects \u003cem\u003eon L. salmonis\u003c/em\u003e infestation in Atlantic Salmon.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll fish handling and procedures were approved by CATC and UPEI Animal Care Committees (AUP 21-008) and carried out under the direct supervision of trained scientists in strict compliance with regulations set out by the Canadian Council for Animal Care (http://www.ccac.ca/). All experiments were performed in accordance with relevant guidelines and regulations, and followed recommendations described by the ARRIVE guidelines for conducting research on animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was part of the \u0026ldquo;CrispResist\u0026rdquo; project funded by the Norwegian Seafood Research Fund (FHF project 901631). DR acknowledges BBSRC Institute Strategic Grants to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A) JEB acknowledges BBSRC Grant to the University of Stirling, BB/V009990/1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the supply of fish (Coho and Pink Salmon) from Quinsam River Hatchery (Paige Ackerman, Edward Walls, Sherri Higgins, Cindy Ederis); Chum Salmon Inch Creek Hatchery (James Wegers); and \u0026Oacute;lafur H. Kristj\u0026aacute;nsson of StofnFiskur, Benchmark Genetics in Iceland and Morten Rye of Benchmark Genetics in Norway for donating and organising the collection and shipment of Atlantic salmon eggs to Canada. Dr. Mark Polinksi and Russell Anderson from FAS delivery for their dedication to fish health and all packaging and shipping of animals; and CATC staff for maintenance of animals throughout the study, Silja Gulliksen for trimming of tissue, and finally David Speare for valuable comments and suggestions.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBajema, I.M., Bruijn, J.A. 2000. What stuff is this! A historical perspective on fibrinoid necrosis. \u003cem\u003eThe Journal of Pathology\u003c/em\u003e, \u003cstrong\u003e191\u003c/strong\u003e(3), 235-238.\u003c/li\u003e\n \u003cli\u003eBraden, L.M., Michaud, D., Groman, D., Byrne, P., Hori, T.S., Fast, M.D. 2023. 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A review of the population biology and host\u0026ndash;parasite interactions of the sea louse Lepeophtheirus salmonis (Copepoda: Caligidae). \u003cem\u003eParasitology\u003c/em\u003e, \u003cstrong\u003e124\u003c/strong\u003e(7), 165-182.\u003c/li\u003e\n \u003cli\u003eZaretsky, J.Z., Wreschner, D.H. 2013. Mucins \u0026ndash; Potential Regulators of Cell Functions. Gel-Forming and Soluble Mucins. \u003cem\u003eBentham Science Publishers Ltd\u003c/em\u003e, \u003cstrong\u003e1\u003c/strong\u003e, 639.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fish skin, spatial transcriptomics, histopathology, mucous cells, L. salmonis, Atlantic Salmon, Chum Salmon, Coho Salmon, Pink Salmon","lastPublishedDoi":"10.21203/rs.3.rs-4947433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4947433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSalmon louse (\u003cem\u003eLepeophtheirus salmonis\u003c/em\u003e) poses significant risks to both wild and farmed salmon populations. Atlantic Salmon (Salmo salar) are highly susceptible to the parasite, while Pacific salmon species such as Pink (\u003cem\u003eOncorhynchus gorbuscha\u003c/em\u003e), Coho (\u003cem\u003eOncorhynchus kisutch\u003c/em\u003e), and Chum (\u003cem\u003eOncorhynchus keta\u003c/em\u003e) salmon exhibit varying levels of resistance. This study explored L. salmonis infestation dynamics in four salmonid species (Atlantic, Pink, Coho, and Chum Salmon) across three challenge trials using the same fish cohort. Infestation was only successful in the third trial, where sedation facilitated parasite attachment. Skin and fin samples were collected at 12, 24, 36, 48, 60, and 168 hours post-infestation (hpi) during the early infestation phase. Results showed low L. salmonis settlement on Chum and Pink Salmon, while Coho Salmon initially had the highest lice counts on fins, followed by Atlantic Salmon. A strong correlation was observed between mucous cell area and susceptibility, with Chum Salmon showing 30-40% mucous cell coverage compared to 10% in Atlantic Salmon. Coho Salmon successfully rejected copepodids after two days, which was linked to an influx of polymorphonuclear cells at attachment sites. This immune response, highlighted by spatial transcriptomics, showed upregulation of host immune markers at the louse-host interface, suggesting that macrophages and neutrophils are key to copepodid rejection. The findings suggest that Coho Salmon’s rapid inflammatory response is crucial in resisting lice and differs significantly from the response in Atlantic Salmon.\u003c/p\u003e","manuscriptTitle":"Local inflammation at the salmon louse (Lepeophtheirus salmonis) attachment site drives copepodid rejection in Coho Salmon (Oncorhynchus kisutch)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-23 16:20:06","doi":"10.21203/rs.3.rs-4947433/v1","editorialEvents":[{"type":"communityComments","content":3},{"type":"decision","content":"Revision requested","date":"2024-11-13T11:32:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-12T13:10:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317426947231879696747431942951985626253","date":"2024-11-02T20:15:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305067629787076356478347616605615403910","date":"2024-11-01T13:08:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-31T18:41:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141567779908927228169034910341782767535","date":"2024-10-29T20:50:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-29T19:14:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155055024245327609538973923070961444342","date":"2024-10-21T17:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225274531652773212422684357620131753172","date":"2024-10-17T17:03:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-17T07:09:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-21T12:23:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-21T12:21:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2024-08-20T21:45:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8be465f1-7f05-42f6-8617-7f0ad92faf1e","owner":[],"postedDate":"September 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:10:18+00:00","versionOfRecord":{"articleIdentity":"rs-4947433","link":"https://doi.org/10.1007/s00441-025-03976-0","journal":{"identity":"cell-and-tissue-research","isVorOnly":false,"title":"Cell and Tissue Research"},"publishedOn":"2025-06-04 15:57:43","publishedOnDateReadable":"June 4th, 2025"},"versionCreatedAt":"2024-09-23 16:20:06","video":"","vorDoi":"10.1007/s00441-025-03976-0","vorDoiUrl":"https://doi.org/10.1007/s00441-025-03976-0","workflowStages":[]},"version":"v1","identity":"rs-4947433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4947433","identity":"rs-4947433","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}