DOI: 10.5061/dryad.gxd254804
title: Data from: Host avoidance and resistance vary independently and are specific to parasite genotype

description: Hosts can reduce the negative fitness effects of parasite infection by
 avoiding contact with parasites or by resisting infection after contact.
 Because of their shared outcome, avoidance and resistance have been
 hypothesized to trade off with one another. Assuming these defenses carry
 fitness costs, hosts are expected to have high levels of one defense or
 the other, but not both. Alternatively, avoidance and resistance may
 covary positively, if, for example, they complement one another or are
 genetically or mechanistically linked. Testing these hypotheses requires
 measuring avoidance and resistance independently, which is challenging
 because they are functionally linked. In this study, we separated
 avoidance and resistance of the host C. elegans against the bacterial
 parasite S. marcescens and tested for a correlation between them. We
 phenotyped a panel of 12 genetically divergent hosts using two distinct
 bacterial strains and multiple experimental contexts. We found no evidence
 of a correlation between avoidance and resistance. This result suggests
 that avoidance and resistance can covary independently. Moreover, we found
 strong genetic specificity not only for resistance, but also for a measure
 of avoidance, motivating further research to examine the coevolutionary
 dynamics of avoidance.
description: Overview We examined the
 relationship between avoidance and resistance to two distinct strains of
 the opportunistic bacterial parasite <em>S. marcescens
 </em>across 12 genetically divergent strains of <em>C.
 elegans</em>. We chose parasite strains that we expected to differ
 in their virulence and the avoidance they elicit. We assayed resistance
 independently of avoidance by measuring survival after directly dosing
 hosts in liquid, which prevents hosts avoiding the parasite. We used two
 assays to measure avoidance: a lawn-leaving assay (Pradel et al., 2007)
 and a choice assay (Schulenburg & Müller, 2004). These are two
 common designs used for assessing behavioral responses to bacteria, and we
 expected they would capture different aspects of behavioral variation. We
 used data from these assays to test whether resistance and avoidance
 covaried across <em>C. elegans</em> strains. Each assay was
 conducted in a blocked design, with a different subset of host strains
 tested in each of four to five experimental blocks that were conducted on
 different days. Each block contained all bacterial treatments (see Table
 S1 for block assignments and Supplementary Text – Section S1 for details
 on statistical handling of block effects).
 Strains
 <em>Hosts </em>
 The 12 genetically divergent strains of <em>C. elegans
 </em>were acquired from the <em>Caenorhabditis
 </em>Natural Diversity Resource (CaeNDR, https://caendr.org/). The
 strains were as follows: CB4856, CX11314, DL238, ED3017, EG4725, JT11398,
 JU258, JU775, LKC34, MY16, MY23, and N2. We assigned host strains a number
 label from 1 to 12 to improve interpretation across the figures (see Table
 S1). CaeNDR recommends using this set of strains as a starting point to
 determine whether genetic variation exists in ecologically relevant traits
 across the species (Cook et al., 2017). In a previous study, these strains
 showed heritable variation in avoidance behavior (lawn-leaving, see below)
 of the <em>S. marcescens</em> strain Db10 (Amoroso et al.,
 2024). Upon receipt of the strains, we followed the
 CaeNDR protocols for cleaning the strains (see
 https://caendr.org/data/protocols), and we cryopreserved them at −80°C.
 Before experiments, we thawed the strains and maintained them on ample
 food (<em>Escherichia coli</em> strain OP50) for 14–17 days at
 20°C prior to an assay to allow populations to recover from
 freezing.
 <em>Parasites</em>
 The two<em> S. marcescens</em> strains used in this
 experiment were Sm2170 and Db10. Sm2170 is a red-pigmented strain that is
 known to be highly virulent, killing the greatest proportion of hosts
 among a set of five <em>S. marcescens </em>strains in a
 previous study (Schulenburg & Ewbank, 2004). Prior studies found
 that this parasite strain is sufficiently virulent to select for avoidance
 and resistance in the host (Morran et al., 2011; Penley et al., 2017,
 2018; Penley & Morran, 2018). The second strain, Db10, is
 unpigmented and elicits a high degree of avoidance from <em>C.
 elegans</em> (Amoroso et al., 2024; Pradel et al., 2007). The
 virulence of Db10 and Sm2170 has not been directly compared, but Db10
 appears to be less virulent (Pradel et al., 2007). Stocks of the bacterial
 strains were maintained at -80ºC in glycerol and thawed for proliferation
 prior to each experiment. Experimental
 Design <em>Quantifying
 Resistance</em> To isolate resistance
 (post-contact defenses) from avoidance (pre-contact), we measured
 infection outcomes after controlling for contact, i.e., by directly dosing
 the host with a known concentration of bacterial cells. We accomplished
 this in a liquid environment, where hosts indiscriminately ingested a
 suspension of the parasite. Then we monitored survival over the subsequent
 days (Schulenburg & Ewbank, 2004; Schulenburg & Müller,
 2004). We used two doses of each of the two parasite strains to maximize
 the potential variation in resistance that we could capture across the 12
 host strains (Fig. 1A). For each <em>C.
 elegans</em> strain, L4 larval stage nematodes were individually
 sorted into wells of a 96-well plate using a Union Biometrica Large
 Particle Flow Cytometer (WormSorter) that initially contained 50 µl of
 sterile S-medium. Each well was inspected microscopically to confirm the
 presence of one live nematode. We excluded wells with more than one
 nematode or a dead nematode. Then the hosts were dosed with an additional
 50 µl of one of five treatments: a high or a low dose of Sm2170, a high or
 a low dose of Db10, or a control dose of OP50. The high and low doses were
 prepared by pelleting 24-hour LB cultures of the two parasite strains
 (initial bacterial concentrations approximately 3.99 +/- 0.43 x
 10<sup>8</sup> CFU/ml for Sm2170; 4.29 +/- 1.2  x
 10<sup>8</sup> CFU/ml for Db10) and resuspending them in
 one-eighth (high dose) or one-quarter (low dose) volume of S-medium. When
 added to the 50 µl of S-medium already in the wells, the high dose was
 concentrated 4x from the initial culture’s cell counts, and the low dose
 was concentrated 2x. For the OP50 control, a 24-hour culture in LB was
 pelleted and resuspended in an equal volume of S-medium. Because it
 included no nutrients, S-medium did not promote additional growth of the
 bacteria in the wells over the course of the experiment. Host strains were
 assigned to wells column-wise in a balanced design across four to five
 plates, and bacterial strain doses were administered row-wise (see Figure
 S1 for details). For each combination of host strain, parasite strain, and
 treatment, the WormSorter filled on average 28.8 +/- 7.45 wells with a
 single L4-stage host. Each plate was covered with Breathe-Easy film and
 incubated at 20ºC, shaking at 160 rpm to promote gas exchange in the
 liquid. After 48 hours, we inspected each well with an
 inverted microscope and scored individual hosts as alive if there was any
 sign of movement (Schulenburg & Müller, 2004). We also recorded
 whether larvae were present in the well, indicating that the host had
 successfully reproduced. Individuals that were missing at 48 hours were
 excluded. We obtained survival data for a mean of 27.6 +/- 7.5 individuals
 per combination of host strain, parasite strain, and treatment, ranging
 from a minimum of 10 to a maximum of 46 individuals per
 combination. <strong> </strong>
 <em>Quantifying Avoidance with a Lawn-leaving
 Assay</em> We first measured avoidance
 using a lawn-leaving assay (Fig. 1B), which is modeled after past studies
 of avoidance of strain Db10 (Amoroso et al., 2024; Pradel et al., 2007).
 We prepared the lawns by pipetting 30µL of 6-hour LB cultures of Db10 or
 Sm2170 onto the center of 60-mm Petri dishes of nematode growth medium
 (NGM-Lite, US Biological, Catalog No: N1005) with 0.7% agarose added to
 discourage <em>C. elegans’</em> burrowing. Using an
 inoculating loop, lawns were spread to cover a 25-mm-diameter circle that
 had been drawn on the bottom of the Petri dish with a template to
 standardize the lawn’s size, shape, and location. The plates were
 incubated overnight at 28ºC. Synchronized populations
 of nematodes were raised on OP50 for 48 hours at 20ºC until they reached
 the L4 larval stage. They were washed from the plates and into a tube with
 M9. They were washed three times with 2 ml of M9 to remove OP50, and then
 approximately 20 individuals were transferred in ~3 µl of M9 to the center
 of the bacterial lawn using a pipette. The precise number of hosts added
 was immediately confirmed by microscopy. After hosts
 were added, the plates were maintained at 20ºC. After 18 and 24 hours, we
 counted the number of individuals located on and off the lawn of bacteria.
 Nematodes touching any part of the lawn were counted as on the lawn. The
 baseline expectation for non-parasitic bacteria is that nearly all
 individuals would be located on the lawn of bacteria at this timepoint, as
 demonstrated by the control treatment in a previous study (Amoroso et al.,
 2024). This experimental protocol follows that of the previously published
 study (Amoroso et al., 2024), except that the transfer of nematodes was
 done in liquid, which is more efficient than picking individual
 nematodes. <strong> </strong>
 <em>Quantifying Avoidance with a Choice Assay
 </em> We also measured behavioral
 responses to <em>S. marcescens</em> in a binary choice assay
 (Fig. 1C), which is a common experimental design for studying <em>C.
 elegans</em>’ bacterial preference (Glater et al., 2014; White et
 al., 2019; Zhang et al., 2005). Here, we grew cultures of Db10 and Sm2170
 in LB for 6 hours. We also grew a culture of OP50 overnight. Onto each
 100-mm Petri dish containing nematode growth medium with 0.7% agarose, we
 added two 10-µl spots of bacterial culture: one spot of OP50, and the
 other spot of either Db10 or Sm2170. The bacterial spots were added on
 opposite sides of the dish, each approximately 1 cm from the inner wall of
 the dish. The dishes were incubated overnight at 28ºC to grow into small
 lawns. <em>C. elegans</em> populations were
 prepared as for the lawn-leaving assay, and approximately 50 L4-stage
 nematodes were added to the center of the agar, equidistant from the two
 bacterial spots. An initial count of nematodes was recorded within the
 first 10 minutes of plating. The number of nematodes located on each lawn
 and on neither lawn was counted at 3 hours, 21 hours, and 27 hours after
 plating. Data Analysis
 Analyses were run in R (v. 4.4.1) (R Core Team, 2023) using
 RStudio (v 2024.04.2+764) (Posit Team, 2023). Data were processed and
 graphics produced with the package <em>tidyverse</em> (Wickham
 et al., 2019). We first used the liquid resistance data
 to examine the effects of each parasite strain on survival compared to the
 OP50 control. Survival was coded as binary (1/0). We fit a generalized
 linear mixed model (‘glmer’ function in the package <em>lme4 (Bates
 et al., 2015)</em>) with a binomial family that included bacterial
 strain and <em>C. elegans</em> strain as fixed effects, and
 plate as a random effect. To examine variation in resistance to the
 parasites across host strains, we fit a second binomial GLMM to the data
 only for the parasite treatments, with host strain, parasite strain, and
 dose as fixed effects. We controlled for plate with a random
 effect. We then used the avoidance data to examine
 variation across host and parasite strains. For the lawn-leaving data, we
 fit a binomial generalized linear mixed model to the counts of hosts on
 and off the parasite lawns, using host strain, parasite strain, and count
 time as predictors, and including a random effect for plate. For the
 choice assay, we calculated a ‘choice index’ for each plate by subtracting
 the number of hosts on the <em>S. marcescens</em> lawn from
 the number on the OP50 lawn and dividing by the total, including hosts on
 neither lawn. A negative choice index indicates attraction to the
 parasite, and a positive choice index indicates avoidance. We fit a linear
 mixed model that included host strain, parasite strain, count time, and
 block as fixed effects, and plate as a random effect.
 Model fits were assessed with the ‘check_model’ function and its
 extensions in the package <em>performance </em>(Lüdecke et
 al., 2021) and tested for overdispersion where appropriate. We estimated
 marginal means (‘emmeans’ function from the package
 <em>emmeans</em> (Lenth, 2023)) and where appropriate,
 converted them from log-odds to percentages for ease of interpretation
 (‘plogis’ function). Models were compared using AIC scores (‘AIC’ and
 ‘compare_performance’ functions). We took two
 approaches to examine correlations between avoidance and resistance. The
 first was to run pairwise correlations (‘cor’ function) between all the
 avoidance and resistance trait variables to determine whether any
 individual measures of avoidance and resistance were correlated. We
 performed a Bonferroni adjustment to correct for multiple comparisons. The
 second was to conduct a principal components analysis (PCA) on the
 avoidance traits and a separate PCA on the resistance traits to capture a
 single axis of variation within each defense category (‘princomp’
 function). We then fit a linear model to the major axis for avoidance and
 the major axis for resistance. For strain-level estimates of each defense
 trait, we took the mean trait value across replicates for each host strain
 for each treatment, and logit-transformed the variables that were measured
 as proportions.
description: # Data from: Host avoidance and resistance vary independently and are
 specific to parasite genotype Dataset DOI:
 [10.5061/dryad.gxd254804](10.5061/dryad.gxd254804) ## Description of the
 data and file structure This study examined the correlation between
 avoidance and resistance of the nematode host *Caenorhabditis elegans* to
 the bacterial parasite *Serratia marcescens*. We measured avoidance with
 two behavioral assay: a lawn-leaving assay and a choice assay. We measured
 resistance as survival in liquid culture with the parasite. The host
 strains were assigned a numeric label to facilitate interpretation of the
 figures in the main text. This is the assignment of labels to host
 strains, which can also be found in the electronic supplementary
 materials. | Label | Host strain | | :---- | :---------- | | 1 | DL238 | |
 2 | MY16 | | 3 | JT11398 | | 4 | N2 | | 5 | EG4725 | | 6 | CB4856 | | 7 |
 CX11314 | | 8 | MY23 | | 9 | ED3017 | | 10 | JU775 | | 11 | LKC34 | | 12 |
 JU258 | ### Files and variables #### File:
 Data_Host_avoidance_and_resistance_vary_independently.zip
 **Description:** Inside of this zipped folder are the files containing the
 data for the analysis. Details follow on the contents of each data file. 
 ``` |-- Data_Host avoidance and resistance vary independently (folder) |--
 |--Choice_assay.csv |-- |--Lawn-leaving_assay.csv |--
 |--Resistance_assay.csv |-- |--Data for supplement (folder) |-- |--
 |--Resistance_assay 24.csv ``` #### **File: Choice_assay.csv**
 Description: Contains data from the choice assay, where groups of C.
 elegans moved from empty agar to one of two patches of bacteria, either E.
 coli strain OP50 or S. marcescens strain Db10 or Sm2170 (depending on the
 treatment). The data file contains the following columns: **Block:** a
 factor assigned to the week during which the experiment was conducted. The
 choice assay was conducted across four experimental blocks, i.e., the data
 were collected on four separate occasions. **Plate_ID:** A unique
 identifier assigned to the replicate that allowed controlling for the
 effect of a single petri dish replicate across multiple time points. 
 **Count_time:** Individual hosts were counted at 3 hours (3h), 21 hours
 (21h), and 27 hours (27h) after being plated.  **Host_strain:** One of
 twelve strains in the "divergent set" from
 CaeNDR [https://caendr.org/request-strains/](https://caendr.org/request-strains/) . In the figures we use numbers to identify the strains for ease of interpretation; see below.  **Bacterial_strain:** Either Sm2170 or Db10, two strains of *S. marcescens*. This is the patch of bacteria on the opposite side of the petri dish from the *E. coli* OP50.  **Worms_on OP50:** Count of individuals on the *E. coli* OP50 patch **Worms_on Smar:** Count of individuals on the *S. marcescens* patch, either Db10 or Sm2170 **Worms_on neither:** Count of individuals on the empty agar. **Choice Index:** Calculated by subtracting the number of hosts on the *S. marcescens* lawn from the number on the *E. coli* OP50 lawn and dividing by the total #### **File: Lawn-leaving_assay.csv** Description: Contains data from the lawn-leaving assay, where individual hosts were added to a circular patch of either *S. marcescens* Sm2170 or Db10. The number of hosts staying on the lawn or leaving was counted the next day.  **Block:** a factor assigned to the week during which the experiment was conducted. The lawn-leaving assay was conducted across five experimental blocks, i.e., the data were collected on five separate occasions. **Plate_ID**: A unique identifier assigned to the replicate that allowed controlling for the effect of a single petri dish replicate across multiple time points.  **Host_strain:** One of twelve strains in the "divergent set" from CaeNDR [https://caendr.org/request-strains/](https://caendr.org/request-strains/) . In the figures we use numbers to identify the strains for ease of interpretation; see below.  **Bacterial_strain:** Either Sm2170 or Db10, two strains of *S. marcescens*. This is the patch of bacteria on the opposite side of the petri dish from the *E. coli* OP50.  **Count_time:** The time that individuals' position was recorded. For this assay, either 18 hours (18h) or 24 hours (24h). **Worms_ON:** Count of *C. elegans* individuals on the patch of bacteria.  **Worms_OFF:** Count of *C. elegans* individuals on the empty agar (avoiding the parasite) **Proportion_avoiding:** This is equal to Worms OFF / (Worms ON + Worms OFF) #### File: Resistance_assay.csv Description: Individual *C. elegans* hosts were added to each well of a 96 well plate and dosed with either *S. marcescens* (eitehr Sm2170 or Db10) or with *E. coli* OP50 in liquid. These results are from 48 hours after the hosts were plated.  **Block:** A factor assigned to the week during which the experiment was conducted. The resistance assay was conducted across five experimental blocks, i.e., the data were collected on five separate occasions. **Host_Strain:** One of twelve strains in the "divergent set" from CaeNDR [https://caendr.org/request-strains/](https://caendr.org/request-strains/) . In the figures we use numbers to identify the strains for ease of interpretation; see below.  **Bacterial_Strain:** Either Sm2170 or Db10, two strains of *S. marcescens*, or the control *E. coli* OP50.  **Dose:** Either High (more concentrated) or Low (less concentrated) doses were given of the two parasite strains. The OP50 control was administered at a single dose. **Alive:** Score of whether the host was moving at all (1) = alive, or no movement detected (0) = dead. **Larvae:** Score of whether L1 larvae were present in the well, indicating that the L4 individual plated two days earlier had successfully reproduced. 1 = larvae present, 0 = no larvae present. **Plate_cont:** A unique identifier assigned to the 96-well plate that controlled for plate effects. It is comprised of the block number, an underscore, and a second number for the plate identity.  **Host_Strain num**: number code from 1 through 12 assigned to the host strains for ease of interpretation in the figures.  #### File: Resistance_assay 24.csv Description: Individual *C. elegans* hosts were added to each well of a 96 well plate and dosed with either *S. marcescens* (eitehr Sm2170 or Db10) or with *E. coli* OP50 in liquid. These results are from 24 hours after the hosts were plated, and results from this dataset are presented in the electronic supplementary materials **Block:** A factor assigned to the week during which the experiment was conducted. The resistance assay was conducted across five experimental blocks, i.e., the data were collected on five separate occasions. **Host_Strain:** One of twelve strains in the "divergent set" from CaeNDR [https://caendr.org/request-strains/](https://caendr.org/request-strains/) . In the figures we use numbers to identify the strains for ease of interpretation; see below.  **Bacterial_Strain:** Either Sm2170 or Db10, two strains of *S. marcescens*, or the control *E. coli* OP50.  **Dose:** Either High (more concentrated) or Low (less concentrated) doses were given of the two parasite strains. The OP50 control was administered at a single dose. **Alive:** Score of whether the host was moving at all (1) = alive, or no movement detected (0) = dead. **Plate_cont:** A unique identifier assigned to the 96-well plate that controlled for plate effects. It is comprised of the block number, an underscore, and a second number for the plate identity.  ## Code/software #### Script: Host_avoidance_and_resistance_vary_independently_Analysis.R **Description:** This is the R script containing the code for the analysis and figures in the main text.  Details on the software and packaged used in this code:  RStudio Version 2025.05.1+513 (2025.05.1+513) R version 4.4.2 (2024-10-31) Platform: aarch64-apple-darwin20 Running under: macOS Sequoia 15.5 Matrix products: default BLAS: /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.0 locale: \[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 time zone: America/New_York tzcode source: internal attached base packages: \[1] stats graphics grDevices utils datasets methods base other attached packages: \[1] performance_0.12.4 optimx_2023-10.21 \[3] ggcorrplot_0.1.4.1 reshape2_1.4.4 \[5] PerformanceAnalytics_2.0.4 xts_0.13.2 \[7] zoo_1.8-12 ggfortify_0.4.17 \[9] lme4_1.1-35.3 Matrix_1.7-1 \[11] emmeans_1.10.1 cowplot_1.1.3 \[13] car_3.1-2 carData_3.0-5 \[15] lubridate_1.9.3 forcats_1.0.0 \[17] stringr_1.5.1 dplyr_1.1.4 \[19] purrr_1.0.2 readr_2.1.5 \[21] tidyr_1.3.1 tibble_3.2.1 \[23] ggplot2_3.5.1 tidyverse_2.0.0 ## Access information N/A