DOI: 10.5061/dryad.0cfxpnw45
title: Population-level variation in parasite resistance due to differences in immune initiation and rate of response

description: Closely related populations often differ in resistance to a given
 parasite, as measured by infection success or failure. Yet, the
 immunological mechanisms of these evolved differences are rarely
 specified. Does resistance evolve via changes to the host’s ability to
 recognize that an infection exists, actuate an effective immune response,
 or attenuate that response? We tested whether each of these phases of the
 host response contributed to threespine sticklebacks’ recently evolved
 resistance to their tapeworm Schistocephalus solidus. While marine
 stickleback and some susceptible lake fish permit fast-growing tapeworms,
 other lake populations are resistant and suppress tapeworm growth via a
 fibrosis response. We subjected lab-raised fish from three populations
 (susceptible marine ‘ancestors’, a susceptible lake population, a
 resistant lake population), to a novel immune challenge using an injection
 of: 1) a saline control, 2) alum, a generalized pro-inflammatory adjuvant
 that causes fibrosis, 3) a tapeworm protein extract, or 4) a combination
 of alum and tapeworm protein). With enough time, all three populations
 generated a robust fibrosis response to the alum treatments. Yet, only the
 resistant population exhibited a fibrosis response to the tapeworm protein
 alone. Thus, these populations differed in their ability to respond to the
 tapeworm protein but shared an intact fibrosis pathway. The resistant
 population also initiated fibrosis faster in response to alum, and was
 able to attenuate fibrosis, unlike the susceptible populations’ slow but
 longer-lasting response to alum. As fibrosis has pathological side-effects
 that reduce fecundity, the faster recovery by the resistant population may
 reflect an adaptation to mitigate the costs of immunity. Broadly, our
 results confirm that parasite detection and immune initiation, activation
 speed, and immune attenuation simultaneously contribute to the evolution
 of parasite resistance and adaptations to infection in natural
 populations.
description: In the spring of 2018, we
 sampled 31 uninfected and 31 infected fish from Gosling Lake and 30
 uninfected and 32 infected fish from Roselle Lake using minnow traps to
 quantify average tapeworm size and the frequency and severity of fibrosis.
 Fish were sampled as part of a larger study where we sampled the first 30
 uninfected fish and then continued sampling until we had found 30 infected
 fish for each population. Fish were categorized as uninfected if we did
 not find a living tapeworm. We scored fibrosis in the peritoneal cavity
 visually using a dissecting microscope as: 0 (no fibrosis), 1 (some
 fibrosis, organs do not move freely), 2 (fibrosis adhering organs
 together), 3 (organs adhered together and to the peritoneal wall), 4
 (severe fibrosis, difficult to open peritoneal cavity) (see supplemental
 video).  We weighed
 tapeworms on a digital scale; tapeworms weighing less than 0.01g were
 recorded as &lt;0.01g (limit of our field scale) and were entered as
 0.009g for summary statistics. If fish were infected with multiple
 tapeworms, we weighed all tapeworms together to get average parasite mass.
 We compared infection intensity between lakes using a general linear model
 (glm) with a Poisson distribution, and average tapeworm mass using a glm
 with a gamma distribution and inverse link function. We also compared the
 number of tapeworms above and below the threshold of our field scale per
 lake using a chi-square test. We compared the fibrosis scores of
 uninfected and infected fish between lakes using Mann-Whitney U tests. To
 get an additional estimate of infection prevalence for Roselle, we
 euthanized and preserved 169 randomly selected fish in ethanol, which were
 later dissected and scored as infected or uninfected (ethanol preservation
 is not conducive to scoring fibrosis). In June 2018, we collected fish
 from our three populations for breeding. Using standard in-vitro
 fertilization methods, we created full-sibling families from each
 population and transported fertilized eggs to the lab for rearing (Divino
 & Schultz 2014). Fish were in two rooms at the animal care
 facility of the University of Connecticut. Families were often, though not
 always, split across multiple tanks located in both rooms. All fish were
 ~11 months old when they were injected with different immune challenges in
 May 2019. <i>Laboratory Injection
 Experiment</i> We injected four different
 inoculants directly into the peritoneal cavity. These included 1)
 20mL of 1X phosphate
 buffered saline (PBS, control treatment), 2) 10mL of homogenized
 tapeworm protein solution + 10mL PBS (tapeworm
 treatment), 3) 10mL of Alum (2% Alumax
 Phosphate, OZ Bioscience)<i> </i>+ 10mL PBS (alum
 treatment), and 4) 10mL tapeworm protein +
 10mL Alum (tapeworm +
 alum treatment). Alum is an immune adjuvant that causes the recruitment of
 leukocytes that initiate an immune response (Kool <i>et
 al.</i> 2012). Pilot studies demonstrated that alum injections could
 induce a fibrosis response in the peritoneal cavity of stickleback
 (<i>Dr. Natalie Steinel, per. comm). </i>The tapeworm protein
 solution was used to test if fish could recognize and respond to tapeworm
 antigens. By using homogenized tapeworms, our experimental design
 mitigates active interference by the tapeworm, which are well known to
 secrete a suite of immunomodulatory molecules that suppress and shift host
 immune responses (Coakley <i>et al.</i> 2016; Maizels
 <i>et al.</i> 2018; Motran <i>et al.</i> 2018),
 including in stickleback (Scharsack <i>et al.</i>
 2013).  To create
 this solution we used tapeworms collected from Farwell Lake (50°11’60”N,
 125°35’27”W) on in 2008 that were flash frozen and stored at -80°C. We
 chose tapeworms from a different lake, watershed, and year to minimize any
 localized genetic structure of the parasite that may influence population
 level responses. Tapeworms were dipped in deionized water and placed in
 chilled 0.9x PBS. Each tapeworm was sonified on ice twice for 1 min
 (Branson 150 Ultrasonic Cell Disruptor, level 5). Between sonification
 rounds, samples were chilled on ice (5 min). The homogenized solutions
 were centrifuged (4°C, 4000rpm, 20min) and the supernatant was collected
 and pooled. We measured the protein concentration using a Red 660 kit
 (G-Biosciences), diluted the solution to 1mg/ml using 0.9x PBS and stored
 it at -20°C.  Before injection, fish were lightly
 anesthetized using a neutral-buffered MS-222 (50-75 mg/L)<i>.
 </i>We used ultra-fine syringes (BD 31G 8mm) to inject 20ul into the
 lower left side of the peritoneal cavity, slightly above where the end of
 ventral spine rests. Injections were shallow and at an angle parallel to
 the body to avoid injuring organs. We watched for visual distention of the
 peritoneal cavity to ensure solutions were being injected correctly.
 Solutions were prepared and syringes were loaded in a sterile culture
 hood. Fish were also given a small colored elastomer mark (Northwest
 Marine Technologies) corresponding to their treatment group injected
 subcutaneously just posterior to the neurocranium. While injecting
 elastomers can impact immune responses (Henrich <i>et al.</i>
 2014), all fish received the same amount of elastomer in the same location
 across treatment groups.<i> </i>During the injection
 procedure, fish were placed on a wet sponge and had their gills covered
 with a wet paper towel. In total, the procedure lasted less than one
 minute, and fish were immediately placed in an aerated recovery tank
 before being returned to their home tank with negligible mortality and no
 noticeable adverse effects. We euthanized fish to measure
 fibrosis post injection at four time points: 1, 10, 42, and 90 days. These
 timepoints were chosen to see 1) if fish could respond within 24 hours to
 an immune challenge, which is when the tapeworm penetrates the gut
 (Hammerschmidt & Kurtz 2007), 2) how their response changed
 through time as the tapeworm would be growing within the peritoneal cavity
 (10 and 42 days). The 10 and 42 day timepoints also correspond to
 timepoints used in previous experiments (Scharsack <i>et
 al.</i> 2007; Weber <i>et al.</i> 2017a, b; Fuess
 <i>et al.</i> 2021), and 3) how this response might change
 within the timeframe of a breeding season, around 90 days. We used the 0-4
 fibrosis scale described above. Two people (AKH & LEF) blind to
 treatment and population scored fibrosis (each fish was scored once). We
 also recorded fish mass, length, and sex. To the best of our ability, we
 spread treatments and time points across families within each population;
 sample sizes are provided in table 1. Sample sizes are small for the 90
 day timepoint as this was added to take advantage of excess surviving
 fish. Throughout the experiment, there was a mortality rate of 11% (48 out
 of 418 fish), which did not appear to be driven by treatment. Mortality
 typically occurred days to weeks after injection.
description: Included in this respository is all code used for the manuscript
 and all datafiles. For more information about fibrosis scores in fish,
 please see manuscript and accompanying video. All analysis were done in R.
 Please contact Amanda Hund with additional questions
 (ahund@umn.edu).