DOI: 10.5061/dryad.7h44j0zt6
title: Inference of symbiotic adaptations in nature using experimental evolution

description: Microbes must adapt to the presence of other species, but it can be
 difficult to recreate the natural context for these interactions in the
 laboratory.  We describe a method for inferring the existence of
 symbiotic adaptations by experimentally evolving microbes that would
 normally interact in an artificial environment without access to other
 species.  By looking for changes in the fitness effects microbes
 adapted to isolation have on their partners, we can infer the existence of
 ancestral adaptations that were lost during experimental
 evolution.  The direction and magnitude of trait changes can
 offer useful insight as to whether the microbes have historically been
 selected to help or harm one another in nature.  We apply our
 method to the complex symbiosis between the social amoeba Dictyostelium
 discoideum and two intracellular bacterial endosymbionts, Paraburkholderia
 agricolaris and P. hayleyella.  Our results suggest P. hayleyella
 – but not P. agricolaris – has generally been selected to attenuate its
 virulence in nature, and that D. discoideum has evolved to
 antagonistically limit the growth of Paraburkholderia.  The
 approach demonstrated here can be a powerful tool for studying adaptations
 in microbes, particularly when the specific natural context in which the
 adaptations evolved is unknown or hard to reproduce.
description: <b>Estimation of
 <i>D. discoideum </i>fitness via spore production
 assays</b> – In order to observe how evolution in isolation affected
 <i>P. agricolaris </i>and <i>P. hayleyella’s
 </i>impact on host fitness, we compared total spore production of
 ancestral <i>D. discoideum </i>strains of interest in the
 presence or absence of ancestral or evolved
 <i>Paraburkholderia</i><b>.</b>  We suspended
 <i>D. discoideum </i>spores in KK2 buffer to a concentration
 of 10<sup>6</sup> spores/mL.  We suspended <i>K.
 pneumoniae </i>and suspended ancestral or evolved
 <i>Paraburkholderia </i>strains in KK2 buffer at an
 OD<sub>600</sub> of 2.0.  We inoculated SM/5 plates with 100uL
 of the <i>D. discoideum </i>suspension
 (~10<sup>5</sup> spores) and 500uL of either 100% <i>K.
 pneumoniae </i>suspension or a 95:5 (vol:vol) mixture of <i>K.
 pneumoniae </i>and <i>Paraburkholderia </i>suspensions. 
 We incubated plates at room temperature under ambient light for 7 days,
 after which we harvested all spores by washing plates with 10mL of a
 detergent solution of 0.1% NP40 in KK2.  We diluted the resulting spore
 suspensions and counted on a hemocytometer to estimate total spore
 production across the entire plate. Spore production assay data is
 contained in the 'sporecountdata.csv' file.  Each line
 represents a single data point - a total spore count from a single agar
 plate, obtained as described above - along with several categorical
 variables to specify which strains, lines, and species
 of <em>Dictyostelium </em>and <em>Paraburkholderia </em>it represents. <b>Estimation of <i>Paraburkholderia </i>fitness via growth rate assays – </b>In order to observe how evolution in isolation affected <i>D. discoideum’s </i>impact on <i>Paraburkholderia</i> fitness, we compared the maximum specific growth rate of ancestral <i>Paraburkholderia </i>strains in liquid culture in the presence or absence of ancestral or evolved <i>D. discoideum</i>.  We inoculated <i>Paraburkholderia </i>strains into liquid SM/5 media and grew them to stationary phase overnight at 30°C in a shaking incubator.  We quantified bacterial suspensions using a spectrophotometer and diluted them to an OD<sub>600</sub> of 0.1.  We prepared <i>D. discoideum </i>amoeba suspensions by inoculating SM/5 plates, incubating them for ~48 hours, and harvesting amoebae by washing plates with 10mL KK2.  We centrifuged amoeba suspensions and washed them with KK2 3-6 times to remove weakly-associated <i>K. pneumoniae, </i>then resuspended them in KK2 buffer containing 300ug/mL tetracycline for 1 hour.  We then centrifuged and washed the antibiotic-treated suspensions 2 more times with fresh KK2 to remove residual tetracycline, counted them using a hemocytometer, and diluted them to 10<sup>7</sup> amoebae/mL.  We obtained growth curves from a Tecan Infinite M200 PRO microplate reader.  We prepared wells of a 96 well plate by combining 100uL SM/5 broth with either 10uL OD<sub>600</sub> 0.1 <i>Paraburkholderia</i> suspension or 10uL KK2 and either 10uL 10<sup>7</sup> amoebae/mL tetracycline-treated <i>D. discoideum </i>suspension or 10uL KK2.  We took OD<sub>600</sub> measurements every 15 minutes for 48 hours to produce a growth curve for each well.  We fit growth curves and calculated maximum specific growth rates using the fitr script (https://github.com/dcangst) in R. Growth rate assay data is contained in the 'growthratedata.csv' file.  Each line represents a single data point - a maximum growth rate measured from a growth curve as described above - along with several categorical variables to specify which strains, lines, and species of <em>Dictyostelium </em>and <em>Paraburkholderia </em>it represents.  Also included is the 'growthratedata_unprocessed.csv' file, which includes the growth curves in their entirety.  Maximum growth rates were determined from these data using the fitr script as described in the accompanying R script file.
description: See comments in R scripts for usage instructions.
 NOTE:  Data files make use of categorical variables
 'Bclade', 'Bstrain', 'Bline', and
 'Bstatus', but the published manuscript instead calls them
 'Pclade', 'Pstrain', 'Pline', and
 'Pstatus'.  This is the result of <em>Paraburkholderia
 agricolaris </em>and <em>P. hayleyella </em>being
 reassigned from the
 genus <em>Burkholderia </em>to <em>Paraburkholderia </em>during the study.  They are referring to the same organisms.