DOI: 10.5061/dryad.b5mkkwhs6
title: Data from: Evidence for the evolution of resistance to non-chemical parasite controls: salmon lice from submerged cages produce larvae that swim deeper

description: Salmon lice (Lepeophtheirus salmonis) pose a major challenge to the
 sustainability of salmon aquaculture due to their capacity to rapidly
 evolve resistance to parasite control methods. As the effectiveness of
 chemical treatments has declined, the industry has increasingly relied on
 preventive strategies to limit initial infections. One such approach is
 depth-based farming, where fish are held deeper in the water column using
 submerged cages. These systems reduce exposure to lice, which typically
 concentrate near the surface. However, there is growing concern that such
 practices may inadvertently select for lice that are better adapted to
 deeper swimming, potentially enabling resistance to depth-based
 interventions. In this study, we investigated whether vertical swimming
 behaviour in salmon lice larvae is influenced by the depth at which their
 parents were collected. We sampled the first generation of 122 adult
 female lice carrying egg strings from commercial salmon farms using either
 standard cages (0–20 m) or submerged cages (20–40 m). Larvae were reared
 under controlled conditions, and the vertical positioning of 11,291
 copepodid larvae was tested in pressure columns simulating a depth of 10
 m. Our results revealed a significant interaction between larval depth
 distribution and the cage type from which the parental lice were sourced
 (χ² = 278.85, df = 1, p &lt; 0.001). Larvae from standard cages showed
 a greater tendency to ascend (35% vs. 23%) and were less likely to sink
 (19% vs. 27%) compared to larvae from submerged cages. These findings
 suggest that vertical swimming behaviour may be heritable, with submerged
 cages potentially selecting for deeper-dwelling lice over time. This study
 provides the first evidence that the depth preference of salmon lice
 larvae may be influenced by their parents’ environment. Understanding this
 behavioural inheritance is crucial for evaluating the long-term
 sustainability of submerged cage systems and for developing lice
 management strategies that anticipate evolutionary responses.
description: <em><strong>Louse Collection and Larval
 Production</strong></em> From early April
 to late May 2024, we collected female <em>Lepeophtheirus
 salmonis</em> from five salmon farms during routine lice counts
 within all cages at the sites (<em>Fig. 1</em>). We gathered
 25 pairs of egg strings from adult females at two standard Atlantic salmon
 cages in Smørdalen and Brattavika, and 22 pairs from a standard cage in
 Uforø. For the submerged farms at Gjengane and Hestabyneset, we collected
 lice from recently deceased fish, as live fish sampling before harvest is
 difficult in submerged technology. The dead fish sank to the bottom of the
 submerged cages, where a lift-up suction pump brought them to the surface.
 The fish were then placed into a bin with a grid to allow water drainage,
 making it easy to collect still-attached lice. We obtained 25 pairs of egg
 strings from Gjengane and 11 pairs of egg strings from Hestabyneset, as
 well as 14 females and 4 males for laboratory reproduction.
 After collection, lice were transported in chilled seawater to
 the Institute of Marine Research’s station in Matre, Masfjorden. Upon
 arrival, egg strings were carefully removed from the lice using tweezers.
 Each pair of egg strings from a female adult was incubated separately
 under controlled conditions (13 °C, 34 PSU), with daily monitoring of
 development. 12 salmon of about 400 g, approximately
 two years old and from the Aquagen strain, were sourced from holding tanks
 at the station. These salmon were placed in 400 L tanks at the research
 station and maintained at 12 °C and 34 PSU for two months. Adult lice
 (both male and female) from submerged cages at Hestabyneset were
 transferred onto these salmon under controlled conditions. The fish were
 sedated with Metomidate Hydrochloride (0.01 g/L, C₁₃H₁₅ClN₂O), and the
 lice were applied to the fish’s skin using tweezers and waterproof paper.
 The lice were monitored until new egg strings were produced. Once this
 occurred, the salmon were sedated again, and the egg strings were
 carefully removed and incubated. All egg strings, whether collected
 directly from farms or produced in the lab, were incubated under the same
 controlled conditions (13 °C, 34 PSU) until they developed into
 copepodids. In total, egg strings from 122 individual females were used in
 the pressurised column experiments: 108 collected directly from standard
 and submerged farms, and 14 produced in the lab from adult lice collected
 at Hestabyneset (submerged cage). These additional egg strings were
 produced to supplement the larvae from Hestabyneset, where egg string
 availability was insufficient. For the experiments, we
 used three-day-old copepodids, the stage at which they are most infectious
 (Tucker et al., 2000; Skern-Mauritzen <em>et al.,</em> 2020).
 Due to natural variation in level of egg string maturity, the copepodids
 hatched in a staggered manner. In total, 11,291 copepodids were assayed to
 investigate their vertical distribution in response to hydrostatic
 pressure, with a focus on how their swimming behaviour might be influenced
 by the parental generation, either from standard or submerged
 cages. <em><strong>Experimental
 Columns</strong></em> Experiments were
 conducted using small-scale water columns, as illustrated in the schematic
 diagram in Fig 1, following procedures established by Coates <em>et
 al.</em> (2020) and Crosbie <em>et al.</em> (2019,
 2020). Each column consisted of a clear polyvinyl chloride (PVC) tube, 85
 cm in height and 4.5 cm in internal diameter, positioned vertically and
 sealed at the bottom. Prior to each experiment, the tube was filled with
 seawater to a height of 80 cm, with a salinity of 34 PSU. The tube was
 then placed inside a transparent PVC box (85 cm x 20 cm x 20 cm) acting as
 a water bath maintained at a constant temperature of 13 °C. A white
 backdrop behind the column was used in order for there to be enough
 contrast to be able to see the copepodids clearly with the naked eye. To
 minimise light exposure, the area was surrounded by black plastic panels,
 keeping the experimental space dark. A PVC lid (28 cm²) was securely
 fastened over the apparatus with bolts, creating a 5 cm air pocket above
 the water. Rubber O-rings ensured an airtight seal. A 6 mm hose, inserted
 through a hole in the lid, connected the column to a scuba tank of
 compressed air via a pressure reducer and adjustable regulator (Festo
 LFR-D-MINI-A, Festo, Australia). The scuba tank introduced air to
 pressurise the air pocket, increasing the hydrostatic pressure of the
 seawater (Stake & Sammarco, 2003). Desired pressure levels were
 achieved in less than 4 seconds, after which no additional air was
 introduced or lost. To validate the apparatus, an inverted graduated test
 tube containing an air bubble was submerged in the column. When 1 bar of
 additional pressure was applied, the air bubble compressed to half its
 original volume, consistent with Boyle’s law.
 <em><strong>Experimental
 assays</strong></em> The swimming behaviour
 of three-day old infectious copepodids (all lice within a single replicate
 came from the same egg string), were tested under 2 atm of pressure
 (ambient pressure at 10 m depth) for a 5-minute interval. This duration
 was chosen based on research by Coates <em>et al.</em> (2020),
 as it captures the maximum behavioural response after which little to no
 further change was observed in the vertical distribution of
 copepodids. Before each experiment, the tube was filled
 halfway with seawater at 34 PSU. Live lice were carefully transferred into
 the tube using a pipette to ensure deceased specimens or any viscous
 residues at the bottom of the incubator were not added. The remaining
 volume of the tube was then filled with seawater, allowing the lice to mix
 evenly throughout the height of the column. After being transferred, the
 lice were left to acclimatise in darkness for 15 minutes. All columns were
 run in darkness (underwater light levels = 0.006–0.03 µmol m² s¹, measured
 with a LI-COR LI-1500 light sensor, LI-COR Biosciences, Germany). After 5
 minutes of pressurisation, the lice distribution was recorded on a sheet
 of paper stuck to the front of the column with water. With the aid of a
 head torch, larvae were visible to the naked eye. The water level in the
 tube was marked, and the distribution of lice was recorded on the sheet at
 10 cm intervals from 10 cm to 80 cm. The counting was done within 45
 seconds to minimise the effect of the head torch light on the lice
 distribution (Szetey <em>et al.,</em> 2021). The same
 scientist recorded the vertical distribution each time to avoid sampling
 bias. In total, 122 columns were run (25 columns each for 5 locations,
 excluding 3 from one location) over a period of 2 months.
 <strong>Statistical Analyses</strong>
 First, we assessed whether the source of the eggs from lice
 sampled from Hestabyneset (whether collected directly from the submerged
 cage or produced after lice were transferred to the laboratory) influenced
 the results. We tested this using a Mann-Whitney U Test. The interaction
 between depth and egg string source (submerged farm vs. laboratory-reared)
 was not significant (W = 11808, p = 1), and patterns of distribution with
 depth were similar (see Supplementary Fig 1). Larvae from both sources for
 Hestabyneset were included in subsequent analyses. To
 analyse the vertical distribution of copepodid-stage salmon lice larvae,
 we calculated the proportion of larvae observed at each 10 cm depth
 interval within the experimental columns. The distribution was bimodal,
 with higher proportions at the surface (0 - 10 cm; hereafter 10 cm)) and
 near the bottom (70 - 80 cm, hereafter 80 cm), which accounted for 53% of
 the observed lice across trials. Intermediate depths (10 – 70 cm) showed
 minimal variation and were excluded from further analysis (as per Coates
 et al., 2020). A generalised linear model (GLM) with a
 binomial distribution was initially used to test the effect of cage type
 (submerged vs. standard) on vertical swimming behaviour at 10 cm and 80
 cm. However, model diagnostics revealed significant overdispersion
 (dispersion ratio = 8.17), indicating that the variance exceeded what a
 standard binomial model could account for. To correct for this, we
 refitted the model using a quasibinomial distribution, which accounts for
 overdispersion and provides more reliable standard errors and significance
 testing. All analyses were performed in R version 4.3.1
 (2023-06-16).
description: # Data from: Evidence for the evolution of resistance to non-chemical
 parasite controls: salmon lice from submerged cages produce larvae that
 swim deeper Dataset DOI:
 [10.5061/dryad.b5mkkwhs6](10.5061/dryad.b5mkkwhs6) ## Description of the
 data and file structure ### Files and variables ### **Column.csv: data
 file** ### **Variables:** * **rep**: Number of replicate column tests
 conducted. Each replicate tests the behaviour of copepods under controlled
 conditions. * **depth**: Depth of the column in centimeters (range: 10–80
 cm in 10cm intervals). * **pressure**: Pressure applied in the column
 (constant at 5 atm) to simulate deeper water conditions. * **lice**:
 Number of lice observed within a specific 10 cm interval of the column. *
 **total.lice**: Total number of lice within a column replicate (sum over
 10–80 cm). * **prop**: Proportion of lice in a 10 cm interval relative to
 the total lice in that column. * **fail**: Number of lice not in the 10 cm
 interval (`total.lice - lice`). * **se**: Standard error of the proportion
 (`prop`). * **col.number**: Column identifier (1–4) indicating which
 column was used and when. * **cagetype**: Type of cage; indicates whether
 larvae originated from standard cages (C) or submerged cages (S). *
 **site**: Farm site from which larvae were collected. * **source**: Origin
 of larvae for submerged cages: either directly collected as egg strings
 from farms or bred in laboratory conditions. **Missing Values:** *
 Represented as `NA`. ## Code/software All analyses were performed in R
 version 4.3.1 (2023-06-16).