DOI: 10.5061/dryad.0rxwdbs2f
title: Pre- and post-association barriers to host switching in sympatric mutualists

description: Coevolution between mutualists can lead to reciprocal specialization,
 potentially causing barriers to host switching.  In the present
 study, we conducted assays to identify pre- and post-association barriers
 to host switching by endosymbiotic bacteria, both within and between two
 sympatric nematode clades.  In nature, Steinernema nematodes and
 Xenorhabdus bacteria form an obligate mutualism.  Free-living
 juvenile nematodes carry Xenorhabdus in a specialized intestinal
 receptacle.  When nematodes enter an insect, they release the
 bacteria into the insect hemocoel.  The bacteria aid in killing
 the insect and facilitate nematode reproduction.  Prior to
 dispersing from the insect, juvenile nematodes must form an association
 with their symbionts; the bacteria must adhere to the intestinal
 receptacle.  We tested for pre-association barriers by comparing
 the effects of bacterial strains on native verses non-native nematodes via
 their virulence towards, nutritional support of, and ability to associate
 with different nematode species.  We then assessed
 post-association barriers by measuring the relative fitness of nematodes
 carrying each strain of bacteria.  We found evidence for both
 pre- and post-association barriers between nematode clades. 
 Specifically, some bacteria were highly virulent to nonnative hosts, and
 some nematode hosts carried fewer cells of nonnative bacteria, leading to
 pre-association barriers.  In addition, reduced infection success
 and lower nematode reproduction were identified as post-association
 barriers.  No barriers to symbiont switching were detected
 between nematode species within the same clade.  Overall, our
 study suggests a framework that could be used to generate predictions for
 the evolution of barriers to host switching in this and other systems.
description: <strong>Data Collection</strong>
   In vitro development assays were
 conducted on two types of media to differentiate bacterial virulence
 towards (LKA media) or nutritional support (LA media) of the nematodes. 
 For each media type, three nematode isolates carrying their native
 symbionts were tested on lawns of 6 strains of bacteria. Five replicates
 of each combination was observed daily for 8 days.  For in vivo
 association assays,we infected each Galleria mellonella caterpillar by
 pipetting 100 nematodes in 500uL of ddH2O on to the dorsum of the
 caterpillar. For pre-association assays, the nematodes were axenic, and
 bacteria cultures were injected into the caterpillars 24 hours after
 nematode infection.  For post-association assays, the nematodes were
 assumed to be carrying bacteria.  Infection success (Infection_Success)
 was measured as the proportion of infected caterpillars with any nematode
 emergence.  Mean nematode emergence (Estimated_IJ_Count) was estimated by
 volumetric subsampling.  We estimated bacterial carriage (CFUs) by
 crushing a sample of 1000 IJs for at least 5 collections per treatment
 group per generation.  If no colonies grew from the crushing, we used the
 detection limit of 0.003 CFU/IJ as the estimated carriage the in
 statistical analyses.    
 <strong>Software and data processing
 description</strong>  
 All in vitro statistical analyses were performed in SAS version
 9.4.  All in vivo statistical analyses were performed in R version
 3.6.3.
description: # Pre- and post-association barriers to host switching in sympatric
 mutualists
 [https://doi.org/10.5061/dryad.0rxwdbs2f](https://doi.org/10.5061/dryad.0rxwdbs2f) ## Description of the data and file structure The data file “JEB in vitro assay dataset.csv” contains 8 columns: A.    Nematode: factor, no unit (Affine, Kraussei): the name of the nematode species used in the infection (*Steinernema affine *or *Steinernema kraussei*) B.    Bacteria: factor , no unit (226, 235, 239, 266, 233, 241) description of the nematode isolate from which the bacteria was cultured.  226 and 235 are *S. affine*, 239 and 266 are *S. kraussei*, 233 and 241 are *S. texanum* C.    Media_Type: factor, no unit (LA, LKA): descriptor of the type of media used for the assay, LA is lipid agar, and is used for the nutrition assay; LKA is liver-kidney agar, and is used for the virulence assay. D.    Day: factor, day (0-8): the census day for each observation. E.    N_Plates: numeric, plates  (1-10): the number of plates exhibiting each nematode developmental stage. F.    Description: factor, no unit (All Dead, Live IJs, Large IJs, Sex Differentiation, Gravid Females, New IJs): Description of each developmental stage observed. G.   Height: numeric, no unit (0-5): The height which corresponds to each developmental stage observed (for graph aesthetics). H.    Prop_Plates: proportion, no unit (0.1-1): The proportion of plates exhibiting each nematode developmental stage. The data file “Assay Fig Lines.csv” contains 6 columns: A.    Nematode: factor, no unit (Affine, Kraussei): the name of the nematode species used in the infection (*Steinernema affine *or *Steinernema kraussei*) B.    Bacteria: factor, no unit (226, 235, 239, 266, 233, 241) description of the nematode isolate from which the bacteria was cultured.  226 and 235 are *S. affine*, 239 and 266 are *S. kraussei*, 233 and 241 are *S. texanum* C.    Media_Type: factor, no unit (LA, LKA): descriptor of the type of media used for the assay, LA is lipid agar, and is used for the nutrition assay; LKA is liver-kidney agar, and is used for the virulence assay. D.    Day: factor, day (0-8): the census day for each observation. E.    Height: numeric, no unit (0-5): The height which corresponds to each developmental stage observed (for graph aesthetics). F.    Group: factor, no unit (1-3): grouping factor for graph aesthetics (required by geom_line) The data file “invitrodataforSAS.csv” contains 10 columns: A.    Nem: factor, no unit isolate code of the nematode species used in the assay 226, 235, or 239 B.    Bact: factor, no unit strain code of bacteria used in the assay (226, 235, 239, 266, 233, 241) C.    Type: factor, no unit media used for the assay, LA is lipid agar, and is used for the nutrition assay; LKA is liver-kidney agar, and is used for the virulence assay. D.    Plate: factor, no unit, identifier for the plate (1-5) E.    Nemcl: factor, no unit, nematode clade, 1 or 3 F.    Bactcl: factor, no unit, nematode clade from which the bacteria were isolated, 1 or 3 G.   Bactsp: factor, no unit, nematode species from which the bacteria were isolated, aff, kra, tex H.    Stage: factor, no unit, Description of developmental stage observed (All Dead, Live IJs, None Visible, Large IJs, Sex Differentiation, Gravid Females, New IJs) I.       Day: factor, day, the census day for each observation (0-8) J.     Stagen: factor, no unit, Developmental stage ordered for logistic regression as 0-6 following the order shown in H.  The data file “JEB in vivo assay dataset.csv” contains 12 columns: A.    Year: numeric, year, description of the batch – 2017 is the first batch, only *S. kraussei *were successfully used; 2018 is the second batch, in which two isolates of *S. affine *and one isolate of *S. kraussei *were used. B.    Generation: numeric, generation, description (0-1) of the passage: 0 is the pre-association assay, in which axenic nematodes were used for infection and bacteria were injected into the caterpillar directly; 1 is the post-association assay, in which nematodes were assumed to be carrying bacteria – no additional bacteria was added. C.    Replicate: numeric, replicate, description (1-2) of the replicate.  If only one replicate was performed, 1 is used.  D.    NemSpp: factor, no unit, (Affine, Kraussei): the name of the nematode species used in the infection (*Steinernema affine *or *Steinernema kraussei*) E.    BactSpp: factor, no unit, (Affine, Affine Control, Kraussei, Kraussei Control, No Bact, Texanum) description of the nematode species from which the bacteria was isolated.  Affine for *S. affine*, Affine Control indicates the unaltered controls – the bacteria was not removed from the nematodes, Kraussei for *S. kraussei*, Kraussei Control indicates the unaltered controls – the bacteria was not removed from the nematodes, No Bact indicates that sterile PBS was injected instead of bacteria cultures, and Texanum, for *S. texanum* F.    Nematode: factor, no unit, (226, 235, 239): description of the nematode isolate used in each infection.  226 and 235 are *S. affine, *and 239 is *S. kraussei* G.   Bacteria: factor, no unit, (226, 235, 239, 266, 233, 241, Control, No Bact) description of the nematode isolate from which the bacteria was cultured.  226 and 235 are *S. affine*, 239 and 266 are *S. kraussei*, 233 and 241 are *S. texanum*, Control indicates the unaltered controls – the bacteria was not removed from the nematodes, and No Bact indicates that sterile PBS was injected instead of bacteria cultures. H.    Host_Mass: numeric, grams, (0.06-0.318) mass of infected caterpillar in grams. I.       Emgd: binary – 1 indicates an infection had nematode emergence, 0 indicates no nematodes emerged from the infection J.     EstIJs: numeric, count, number of juvenile nematodes which emerged from successful infections. n/a indicates that no nematodes were counted. K.    CFUstoLog: numeric, count, (0.0027 – 35.04) bacterial carriage calculated as the number of colony forming units (CFUs) divided by the number of nematodes crushed in the sample, adjusted for dilution.  If no colonies grew, the detection limit, defined as the fewest cells detectable in our study was used instead. n/a indicates that no samples were crushed. L.     LogCFUs: numeric, log count, calculated as log(CFUs). n/a indicates that no samples were crushed. ## Sharing/Access information **Licence and Restriction statement** There are no licences or restrictions placed on access to the dataset or any associated files. ## Code/Software **SAS Code Description** Statistical analyses of the in vitro development assays were performed in SAS 9.4 using the file “invitroSAS.txt”.  To run in SAS, save as a .SAS file or copy the text into the editor window.  The program performs ordered logistic regression, first on the Clade 1, S. affine nematodes, then on the Clade 3, S. kraussei nematodes.   **R Code Description** The statistical analyses were performed in R using the file “JEB_Association Barriers_Dryad Code.Rmd” in R Studio.  The first chunk of code loads libraries and set up options for the run.  This file uses libraries ggplot2 (to generate plots), dplyr (to filter and process data), emmeans (to calculate estimated marginal means), lme4 (for mixed models), car (for type 3 F-tests), afex (to calculate the test of fixed effects in mixed models), and rmarkdown (to generate paged tables).  To simplify the output, I set scipen = 999, to prevent scientific notation of p-values.  I also set contrasts to “contr.sum” or “contr.poly” to accurately calculate the type 3 fixed effects. The second chunk creates aesthetics for the plots, including fill and color palettes, and a central theme. The third chunk reads in two .csv data files for the in vitro assays, and creates the Virulence Assay plots, Figure 2. The  fourth chunk creates the Nutrition Assay plots, Figure 3. The fifth chunk reads in the .csv in vivo assay data file, and subsets the data into pre- and post-association assays for each nematode species used (*S. affine* vs. *S. kraussei*) and for the two batches of *S. kraussei.* The sixth chunk analyses emergence probability for the pre-association assay of *S. affine *nematodes and creates Figure 4A. The seventh chunk analyses mean nematode emergence for the pre-association assay of *S. affine *nematodes and creates Figure 4B. The eighth chunk analyses mean log bacterial carriage for the pre-association assay of *S. affine *nematodes and creates Figure 4C. The ninth chunk analyses emergence probability for the post-association assay of *S. affine *nematodes and creates Figure 4D. The tenth chunk analyses emergence probability for the post-association assay of the first batch of *S. kraussei *nematodes and creates Figure 5A. The eleventh chunk analyses mean nematode emergence for the post-association assay of the first batch of *S. kraussei *nematodes and creates Figure 5B. The twelfth chunk analyses mean log bacterial carriage for the post-association assay of the first batch of *S. kraussei *nematodes and creates Figure 5C. The thirteenth chunk analyses emergence probability for the pre-association assay of the second batch of *S. kraussei *nematodes and creates Figure 5D. The fourteenth chunk analyses mean nematode emergence for the pre-association assay of the second batch of *S. kraussei *nematodes and creates Figure 5E. The fifteenth chunk analyses mean log bacterial carriage for the pre-association assay of the second batch of *S. kraussei *nematodes and creates Figure 5F. The sixteenth chunk analyses emergence probability for the post-association assay of the second batch of *S. kraussei *nematodes and creates Figure 5G. The sevententh chunk analyses mean nematode emergence for the post-association assay of the second batch of *S. kraussei *nematodes and creates Figure 5H. The eighteenth chunk analyses mean log bacterial carriage for the post-association assay of the second batch of *S. kraussei *nematodes and creates Figure 5I.