<html lang="en"> <head> <title>Formicine ants swallow their highly acidic poison for gut microbial selection and control </title> <meta charset="utf-8"> <meta name="viewport" content="width=device-width, initial-scale=1.0"> <meta http-equiv="X-UA-Compatible" content="ie=edge"> <link href="https://unpkg.com/@stencila/thema@2/dist/themes/elife/styles.css" rel="stylesheet"> <script src="https://unpkg.com/@stencila/thema@2/dist/themes/elife/index.js" type="text/javascript"></script> <script src="https://unpkg.com/@stencila/components@<=1/dist/stencila-components/stencila-components.esm.js" type="module"></script> <script src="https://unpkg.com/@stencila/components@<=1/dist/stencila-components/stencila-components.js" type="text/javascript" nomodule=""></script> </head> <body> <main role="main"> <article itemscope="" itemtype="http://schema.org/Article" data-itemscope="root"> <h1 itemprop="headline">Formicine ants swallow their highly acidic poison for gut microbial selection and control</h1> <meta itemprop="image" content="https://via.placeholder.com/1200x714/dbdbdb/4a4a4a.png?text=Formicine%20ants%20swallow%20their%20highly%20acidic%20poison%20for%20gut%20microbial%20selection%20and%20control"> <ol data-itemprop="authors"> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Simon Tragust"><span data-itemprop="givenNames"><span itemprop="givenName">Simon</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Tragust</span></span><span data-itemprop="emails"><a itemprop="email" href="mailto:simon.tragust@zoologie.uni-halle.de">simon.tragust@zoologie.uni-halle.de</a></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Claudia Herrmann"><span data-itemprop="givenNames"><span itemprop="givenName">Claudia</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Herrmann</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Jane Häfner"><span data-itemprop="givenNames"><span itemprop="givenName">Jane</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Häfner</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Ronja Braasch"><span data-itemprop="givenNames"><span itemprop="givenName">Ronja</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Braasch</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Christina Tilgen"><span data-itemprop="givenNames"><span itemprop="givenName">Christina</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Tilgen</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Maria Hoock"><span data-itemprop="givenNames"><span itemprop="givenName">Maria</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Hoock</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Margarita Artemis Milidakis"><span data-itemprop="givenNames"><span itemprop="givenName">Margarita</span><span itemprop="givenName">Artemis</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Milidakis</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Roy Gross"><span data-itemprop="givenNames"><span itemprop="givenName">Roy</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Gross</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-2">2</a></span> </li> <li itemscope="" itemtype="http://schema.org/Person" itemprop="author"> <meta itemprop="name" content="Heike Feldhaar"><span data-itemprop="givenNames"><span itemprop="givenName">Heike</span></span><span data-itemprop="familyNames"><span itemprop="familyName">Feldhaar</span></span><span data-itemprop="affiliations"><a itemprop="affiliation" href="#author-organization-1">1</a></span> </li> </ol> <ol data-itemprop="affiliations"> <li itemscope="" itemtype="http://schema.org/Organization" itemid="#author-organization-1" id="author-organization-1"><span itemprop="name">Animal Ecology I, Bayreuth Center for Ecology and Environmental Research (BayCEER), University of Bayreuth, Universitätsstraße</span><address itemscope="" itemtype="http://schema.org/PostalAddress" itemprop="address"><span itemprop="addressLocality">Bayreuth</span><span itemprop="addressCountry">Germany</span></address></li> <li itemscope="" itemtype="http://schema.org/Organization" itemid="#author-organization-2" id="author-organization-2"><span itemprop="name">Microbiology, Biocenter, University of Würzburg, Am Hubland</span><address itemscope="" itemtype="http://schema.org/PostalAddress" itemprop="address"><span itemprop="addressLocality">Würzburg</span><span itemprop="addressCountry">Germany</span></address></li> </ol><span itemscope="" itemtype="http://schema.org/Organization" itemprop="publisher"> <meta itemprop="name" content="Unknown"><span itemscope="" itemtype="http://schema.org/ImageObject" itemprop="logo"> <meta itemprop="url" content="https://via.placeholder.com/600x60/dbdbdb/4a4a4a.png?text=Unknown"> </span> </span><time itemprop="datePublished" datetime="2020-11-03">2020-11-03</time> <ul data-itemprop="genre"> <li itemprop="genre">Research Article</li> </ul> <ul data-itemprop="about"> <li itemscope="" itemtype="http://schema.org/DefinedTerm" itemprop="about"><span itemprop="name">Evolutionary Biology</span></li> <li itemscope="" itemtype="http://schema.org/DefinedTerm" itemprop="about"><span itemprop="name">Microbiology and Infectious Disease</span></li> </ul> <ul data-itemprop="keywords"> <li itemprop="keywords">Camponotus floridanus</li> <li itemprop="keywords">Acetobacteraceae</li> <li itemprop="keywords">Serratia marcescens</li> <li itemprop="keywords">phylosymbiosis</li> <li itemprop="keywords">microbiom</li> <li itemprop="keywords">external immune defense</li> <li itemprop="keywords">Other</li> </ul> <ul data-itemprop="identifiers"> <li itemscope="" itemtype="http://schema.org/PropertyValue" itemprop="identifier"> <meta itemprop="propertyID" content="https://registry.identifiers.org/registry/publisher-id"><span itemprop="name">publisher-id</span><span itemprop="value" data-itemtype="http://schema.org/Number">60287</span> </li> <li itemscope="" itemtype="http://schema.org/PropertyValue" itemprop="identifier"> <meta itemprop="propertyID" content="https://registry.identifiers.org/registry/doi"> <span itemprop="name">doi</span><span itemprop="value">10.7554/eLife.60287</span> </li> <li itemscope="" itemtype="http://schema.org/PropertyValue" itemprop="identifier"> <meta itemprop="propertyID" content="https://registry.identifiers.org/registry/elocation-id"><span itemprop="name">elocation-id</span><span itemprop="value">e60287</span> </li> </ul> <section data-itemprop="description"> <h2 data-itemtype="http://schema.stenci.la/Heading">Abstract</h2> <meta itemprop="description" content="Animals continuously encounter microorganisms that are essential for health or cause disease. They are thus challenged to control harmful microbes while allowing the acquisition of beneficial microbes. This challenge is likely especially important for social insects with respect to microbes in food, as they often store food and exchange food among colony members. Here we show that formicine ants actively swallow their antimicrobial, highly acidic poison gland secretion. The ensuing acidic environment in the stomach, the crop, can limit the establishment of pathogenic and opportunistic microbes ingested with food and improve the survival of ants when faced with pathogen contaminated food. At the same time, crop acidity selectively allows acquisition and colonization by Acetobacteraceae, known bacterial gut associates of formicine ants. This suggests that swallowing of the poison in formicine ants acts as a microbial filter and that antimicrobials have a potentially widespread but so far underappreciated dual role in host-microbe interactions."> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Animals continuously encounter microorganisms that are essential for health or cause disease. They are thus challenged to control harmful microbes while allowing the acquisition of beneficial microbes. This challenge is likely especially important for social insects with respect to microbes in food, as they often store food and exchange food among colony members. Here we show that formicine ants actively swallow their antimicrobial, highly acidic poison gland secretion. The ensuing acidic environment in the stomach, the crop, can limit the establishment of pathogenic and opportunistic microbes ingested with food and improve the survival of ants when faced with pathogen contaminated food. At the same time, crop acidity selectively allows acquisition and colonization by Acetobacteraceae, known bacterial gut associates of formicine ants. This suggests that swallowing of the poison in formicine ants acts as a microbial filter and that antimicrobials have a potentially widespread but so far underappreciated dual role in host-microbe interactions.</p> </section> <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="introduction">Introduction </h2> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Animals commonly harbor gut-associated microbial communities (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib42">bib42</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib99">bib99</a></cite>). Patterns of recurring gut microbial communities have been described for many animal groups (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib20">bib20</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib84">bib84</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib106">bib106</a></cite>). The processes generating these patterns are however often not well understood. They might result from host filtering (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib94">bib94</a></cite>), a shared evolutionary history between gut-associated microbes and their hosts (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib98">bib98</a></cite>) involving microbial adaptations to the host environment (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib95">bib95</a></cite>), simply be a byproduct of similar host dietary preferences (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib2">bib2</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib56">bib56</a></cite>), or result from interactions between microbes in the gut-associated microbial community (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib15">bib15</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib48">bib48</a></cite>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Food is an important environmental source of microbial gut associates (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib12">bib12</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib16">bib16</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib36">bib36</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib56">bib56</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib115">bib115</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib114">bib114</a></cite>) but also poses a challenge, the need to discriminate between harmful and beneficial microbes, as food may contain microbes that produce toxic chemicals or that are pathogenic (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib24">bib24</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib38">bib38</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib73">bib73</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib142">bib142</a></cite>). In social animals, control of harmful microbes in food while at the same time allowing the acquisition and transmission of beneficial microbes from and with food, is likely especially important. Eusocial Hymenoptera not only transport and store food in their stomach, the crop, but also distribute food to members of their colony via trophallaxis, i.e. the regurgitation of crop content from donor individuals to receiver individuals through mouth-to-mouth feeding (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib49">bib49</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib53">bib53</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib86">bib86</a></cite>). While trophallaxis can facilitate the transmission of beneficial microbes, it can also entail significant costs, as it might open the door to unwanted microbial opportunists and pathogens that can take advantage of these transmission routes (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib108">bib108</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib125">bib125</a></cite>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Here we investigate how formicine ants, specifically the Florida carpenter ant <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em>, solve the challenge to control harmful microbes in their food while allowing acquisition and transmission of beneficial microbes from and with their food. Apart from specialized intracellular endosymbionts associated with the midgut in the ant tribe Camponotini (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib37">bib37</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib45">bib45</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib124">bib124</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib148">bib148</a></cite>), formicine ant species have only low abundances of microbial associates in their gut lumen but carry members of the bacterial family Acetobacteraceae as a recurring part of their gut microbiota (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib18">bib18</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib31">bib31</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib59">bib59</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib72">bib72</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib124">bib124</a></cite>). Some formicine gut-associated Acetobacteraceae show signs of genomic and metabolic adaptations to their host environment indicating coevolution and mutual benefit (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib19">bib19</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib32">bib32</a></cite>). But the recurrent presence of Acetobacteraceae in the gut of formicine ants potentially also reflects the direct transmission of bacteria among individuals, selective uptake on the part of the ants, specific adaptations for colonizing ant guts on the part of the bacteria, or some combination of all three (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib42">bib42</a></cite>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Formicine ant species possess a highly acidic poison gland secretion containing formic acid as its main component (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib88">bib88</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib110">bib110</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib128">bib128</a></cite>). Although the poison is presumably foremost used as a defensive weapon (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib111">bib111</a></cite>), it is also distributed to the environment of these ants as an external immune defense trait (sensu <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib112">bib112</a></cite>) to protect their offspring and the nest and to limit disease spread within the society (see references in <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib141">bib141</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib21">bib21</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib116">bib116</a></cite>). To this end, ants take up their poison from the acidopore, the opening of the poison gland at the gaster tip, into their mouth (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib140">bib140</a></cite>) during a specialized behavior existing only in a subset of ant families among all Hymenopterans (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib5">bib5</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib43">bib43</a></cite>), termed acidopore grooming.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Here we first investigate whether the poison is also swallowed during acidopore grooming in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> and seven other formicine ant species from three genera in a comparative survey. In survival experiments and in in vitro and in vivo bacterial viability and growth experiments, we then investigate whether swallowing of the poison can serve gut microbial control and may prevent bacterial pathogen infection. Complementing these experiments, we also test whether poison swallowing has the potential to limit pathogen transmission during trophallactic food exchange. Finally, we explore whether swallowing of the poison acts as a microbial filter that is permissible to gut colonization by bacteria from the family Acetobacteraceae.</p> <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="results">Results</h2> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#computational packages and code library(plyr)#version 1.8.4 library(dplyr)#version 0.8.3 drop.levels <- function(dat){ # Drop unused factor levels from all factors in a data.frame # Author: Kevin Wright. Idea by Brian Ripley. dat[] <- lapply(dat, function(x) x[,drop=TRUE]) return(dat) } #graphical packages library(ggplot2)#version 3.2.1 library(ggkm)#version 0.6.0; see https://github.com/sachsmc/ggkm #statistical packages library(lme4)#version 1.1-21 library(multcomp)#version 1.4-10 library(DHARMa)#version 0.2.4 library(coxme)#2.2-14 library(glmmTMB)#version 0.2.3 #in order for the package multcomp to work with glmmTMB you need this function see: https://github.com/glmmTMB/glmmTMB/blob/master/glmmTMB/vignettes/model_evaluation.rmd glht_glmmTMB <- function (model, ..., component="cond") { glht(model, ..., coef. = function(x) fixef(x)[[component]], vcov. = function(x) vcov(x)[[component]], df = NULL) } library(broman) # for myround function to round p-values for inline text format_pval <- function(x){ if (x < .001) return(paste('<0.001')) if (x > .05 & x < 0.1) return(paste('>0.05')) paste('=', myround(x, 3)) # 3 = no. of digits to round p value to if .001 < p < .250. } # the raw data is stored at the Dryad Digital Data Repository: https://doi.org/10.5061/dryad.k0p2ngf4v </code></pre> </stencila-code-chunk> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="swallowing-of-the-formicine-ant-poison-gland-secretion-leads-to-acidic-crop-environments"> Swallowing of the formicine ant poison gland secretion leads to acidic crop environments </h3> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>###################################################################################################### ######################### Fig 1 a,b,c: Crop acidification ############################################ ###################################################################################################### #############loading data for Fig.1a acidification.c.floridanus<-read.delim("https://datadryad.org/stash/downloads/file_stream/452657",header=T)#choose file: Supplementary Data Fig.1a.txt #str(acidification.c.floridanus) acidification.c.floridanus$time<-factor(acidification.c.floridanus$time,c("0+4h","0+24h","0+48h","48+4h"))#reorder factor time #############statistcal analysis m.acidification.c.floridanus<-lmer(log(ph)~time+(1|colony),data=acidification.c.floridanus) #summary(m.acidification.c.floridanus) #model validation res.m.acidification.c.floridanus<-simulateResiduals(fittedModel=m.acidification.c.floridanus) #plot(res.m.acidification.c.floridanus) #significance of predictor time since feeding (four levels: 0+4h, 0+24h, 0+48h, 48h+4h) m.acidification.c.floridanus0<-lmer(log(ph)~1+(1|colony),data=acidification.c.floridanus) #anova(m.acidification.c.floridanus,m.acidification.c.floridanus0) #post-hoc comporisons of predictor-levels time since feeding m.acidification.c.floridanus.glht<-glht(m.acidification.c.floridanus,linfct=mcp(time="Tukey")) output.m.acidification.c.floridanus.glht<-summary(m.acidification.c.floridanus.glht,test=adjusted("Westfall")) #output.m.acidification.c.floridanus.glht #calculation of median values and confidence intervals of predictor time since feeding (four levels: 0+4h, 0+24h, 0+48h, 48h+4h) ph.summary.c.floridanus<-ddply(acidification.c.floridanus,"time",summarize, N=length(ph), median=quantile(ph,0.5), lowerCI=quantile(ph,0.025), upperCI=quantile(ph,0.975)) #ph.summary.c.floridanus #############loading data for Fig.1b mobility.c.floridanus<-read.delim("https://datadryad.org/stash/downloads/file_stream/452656",header=T)#choose file: Supplementary Data Fig.1b.txt #str(mobility.c.floridanus) #############statistcal analysis for Fig.1b m.mobility.c.floridanus<-lmer(ph~treatment+(1|colony),data=mobility.c.floridanus) #summary(m.mobility.c.floridanus) #model validation res.m.mobility.c.floridanus<-simulateResiduals(fittedModel=m.mobility.c.floridanus) #plot(res.m.mobility.c.floridanus) #significance of predictor ant manipulation (two levels: FA+ and FA-, i.e. ants with and without acidopore access) m.mobility.c.floridanus0<-lmer(ph~1+(1|colony),data=mobility.c.floridanus) #anova(m.mobility.c.floridanus,m.mobility.c.floridanus0) #############loading data for Fig.1c mobility.formicine.ants<-read.delim("https://datadryad.org/stash/downloads/file_stream/452659",header=T)#choose data: Supplementary Data Fig.1c.txt #str(mobility.formicine.ants) #subsetting the data frame into the six formicine ant species mobility.c.maculatus<-subset(mobility.formicine.ants,colony=="c2") mobility.c.maculatus<-drop.levels(mobility.c.maculatus) #str(mobility.c.maculatus) mobility.l.fuliginosus<-subset(mobility.formicine.ants,colony=="l1") mobility.l.fuliginosus<-drop.levels(mobility.l.fuliginosus) #str(mobility.l.fuliginosus) mobility.f.pratensis<-subset(mobility.formicine.ants,colony=="f1") mobility.f.pratensis<-drop.levels(mobility.f.pratensis) #str(mobility.f.pratensis) mobility.f.rufibarbis<-subset(mobility.formicine.ants,colony=="f2") mobility.f.rufibarbis<-drop.levels(mobility.f.rufibarbis) #str(mobility.f.rufibarbis) mobility.f.cinerea<-subset(mobility.formicine.ants,colony=="f3") mobility.f.cinerea<-drop.levels(mobility.f.cinerea) #str(mobility.f.cinerea) mobility.f.cunicularia<-subset(mobility.formicine.ants,colony=="f4") mobility.f.cunicularia<-drop.levels(mobility.f.cunicularia) #str(mobility.f.cunicularia) mobility.f.fuscocinerea<-subset(mobility.formicine.ants,colony=="f5") mobility.f.fuscocinerea<-drop.levels(mobility.f.fuscocinerea) #str(mobility.f.fuscocinerea) #############statistcal analysis for Fig.1c #wilcox.test(ph~treatment,data=mobility.c.maculatus) #wilcox.test(ph~treatment,data=mobility.l.fuliginosus) #wilcox.test(ph~treatment,data=mobility.f.pratensis) #wilcox.test(ph~treatment,data=mobility.f.rufibarbis) #wilcox.test(ph~treatment,data=mobility.f.cinerea) #wilcox.test(ph~treatment,data=mobility.f.cunicularia) #wilcox.test(ph~treatment,data=mobility.f.fuscocinerea) ###################################################################################################### ###################### Fig. 1 - figure supplement 1: crop acidity baseline ########################### ###################################################################################################### #############loading data for Fig.1 - figure supplement 1 acidification.baseline<-read.delim("https://datadryad.org/stash/downloads/file_stream/452660",header=T)#choose file: Supplementary Data Fig.1 - figure supplement 1.txt #str(acidification.baseline) #calculation of median values and confidence intervals of predictor status (two levels:satiated and starved) and worker caste (two levels: minor and major) ph.summary.acidification.baseline<-ddply(acidification.baseline,c("status","caste"),summarize, N=length(ph), median=quantile(ph,0.5), lowerCI=quantile(ph,0.025), upperCI=quantile(ph,0.975)) #ph.summary.acidification.baseline ###################################################################################################### ###################### Fig. 1 - figure supplement 2: acidopore grooming ############################## ###################################################################################################### #############loading data for Fig. 1 - figure supplement 2 acidopore.grooming<-read.delim("https://datadryad.org/stash/downloads/file_stream/452661",header=T)#choose file: Supplementary Data Fig.1 - figure supplement 2.txt #str(acidopore.grooming) food.treatment1<-factor(acidopore.grooming$food.treatment,levels=c("unfed","water","honey"))#reorder factor treatment #############statistcal analysis for Fig. 1 - figure supplement 2 m.grooming<-glmer.nb(groomingevents~food.treatment+(1|colony),data=acidopore.grooming) #summary(m.grooming) #model validation res.m.grooming<-simulateResiduals(fittedModel=m.grooming) #plot(res.m.grooming) #significance of predictor food treatment (three levels: unfed, water, honey) m.grooming0<-glmer.nb(groomingevents~1+(1|colony),data=acidopore.grooming) #anova(m.grooming,m.grooming0) #post-hoc comporisons of predictor-levels food treatment m.grooming.glht<-glht(m.grooming,linfct=mcp(food.treatment="Tukey")) output.m.grooming.glht<-summary(m.grooming.glht,test=adjusted("Westfall")) #output.m.grooming.glht ###################################################################################################### ############################### Fig. 1 - figure supplement 3: pH midgut ############################# ###################################################################################################### #############loading data for Fig.1 - figure supplement 3 ph.midgut<-read.delim("https://datadryad.org/stash/downloads/file_stream/452662", header=T)#choose data: Supplementary Data Fig.1 - figure supplement 3.txt #str(ph.midgut) #############statistcal analysis for Fig.1 - figure supplement 3 m.ph.midgut<-lmer(ph~gutregion+(1|colony),data=ph.midgut) #summary(m.ph.midgut) #model validation res.m.ph.midgut<-simulateResiduals(fittedModel=m.ph.midgut) #plot(res.m.ph.midgut) #significance of predictor gutregion (five levels: crop, midgut.position1, midgut.position2, midgut.position3, midgut.position4) m.ph.midgut0<-lmer(ph~1+(1|colony),data=ph.midgut) #anova(m.ph.midgut,m.ph.midgut0) #post-hoc comporisons of predictor-levels position m.ph.midgut.glht<-glht(m.ph.midgut,linfct=mcp(gutregion="Tukey")) output.m.ph.midgut.glht<-summary(m.ph.midgut.glht,test=adjusted("Westfall")) #output.m.ph.midgut.glht #calculation of median values and confidence intervals of predictor position ph.summary.midgut<-ddply(ph.midgut,"gutregion",summarize, N=sum(!is.na(ph)), median=quantile(ph,0.5,na.rm=TRUE), lowerCI=quantile(ph,0.025,na.rm=TRUE), upperCI=quantile(ph,0.975,na.rm=TRUE), mean=mean(ph,na.rm=TRUE), sd=sd(ph,na.rm=TRUE), length=length(!is.na(ph)), se=sd/sqrt(length)) #ph.summary.midgut </code></pre> </stencila-code-chunk> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To reveal whether formicine ants swallow their acidic poison during acidopore grooming, we first monitored acidity levels in the crop lumen of the Florida carpenter ant <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em> after feeding them 10% honey water (pH = 5). We found that after feeding the crop became increasingly acidic over time, reaching highly acidic values 48 hr after feeding (median pH = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.c.floridanus[3,3]</code><output slot="output"></output> </stencila-code-expression>; 95% CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(ph.summary.c.floridanus[3,4],1)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.c.floridanus[3,5],1)</code><output slot="output"></output></stencila-code-expression>), whilst renewed access to food after 48 hr raised the pH to levels recorded after the first feeding (<a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1a</a>; LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.acidification.c.floridanus,m.acidification.c.floridanus0)[2,6],2)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.acidification.c.floridanus,m.acidification.c.floridanus0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.acidification.c.floridanus,m.acidification.c.floridanus0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>; Westfall corrected post-hoc comparisons: 0+4 hr versus. 48h+4 hr: p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.acidification.c.floridanus.glht$test)$pvalues[3],3)</code><output slot="output"></output></stencila-code-expression>, all other comparisons: p <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.acidification.c.floridanus.glht$test)$pvalues[2],3))</code><output slot="output"></output></stencila-code-expression>). We also found that crop pH levels of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants were highly acidic in workers taken directly out of a satiated colony (<a href="#fig1s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 1</a>; major workers: median pH = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.acidification.baseline[1,4]</code><output slot="output"></output></stencila-code-expression>, 95% CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[1,5],1)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[1,6],1)</code><output slot="output"></output></stencila-code-expression>; minor workers: median pH = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.acidification.baseline[2,4]</code><output slot="output"></output></stencila-code-expression>, CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[2,5],1)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[2,6],1)</code><output slot="output"></output></stencila-code-expression>) and in worker cohorts that were satiated for 3 d and then starved for 24 hr before measurements (majors: median pH = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.acidification.baseline[3,4]</code><output slot="output"></output></stencila-code-expression>, 95% CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[3,5],1)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[1,6],1)</code><output slot="output"></output></stencila-code-expression>; minors: median pH = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.acidification.baseline[4,4]</code><output slot="output"></output></stencila-code-expression>, CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[4,5],1)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.acidification.baseline[4,6],1)</code><output slot="output"></output></stencila-code-expression>), suggesting that under natural conditions an acidic baseline pH in the crop lumen is maintained following perturbation thereof through ingested fluids.</p> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1a" title="Figure 1a"> <label data-itemprop="label">Figure 1a</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 50 #' @height 35 #addition of median values per time since feeding into the data.frame as a new variable acidification.c.floridanus2<-acidification.c.floridanus%>%group_by(time)%>%mutate(medianph=median(ph)) #definition of universal ph indicator colors: 1-6 ph.colors.rgb1<-c( "202 58 67", "235 81 42", "252 125 19", "253 190 2", "254 250 0", "235 230 54" ) #transformation of RGB colors into hex colors ph.colors.hex1<-sapply(strsplit(ph.colors.rgb1, " "), function(ph.colors.rgb1) rgb(ph.colors.rgb1[1], ph.colors.rgb1[2], ph.colors.rgb1[3], maxColorValue=255)) #plotting of Fig.1a fig.1a<-ggplot()+ geom_boxplot(data=acidification.c.floridanus2,mapping=aes(x=time,y=ph,fill=medianph),colour="grey30",lwd=1.5,outlier.shape=NA,alpha=0.5,width=0.4,coef=0)+ scale_fill_gradientn(colours=ph.colors.hex1,breaks=c(1,2,3,4,5,6),limits=c(1,6.5),guide=FALSE)+ geom_point(data = acidification.c.floridanus,mapping=aes(x=time,y=ph,fill=ph),pch=21,color="white",position=position_jitter(width=0.1),size=6.5,alpha=1)+ scale_x_discrete(name="Time after feeding",labels=c("0h+4h","0h+24h","0h+48h","48h+4h"))+ scale_y_continuous(name="pH-value",limits=c(1,6),breaks=c(1,2,3,4,5,6))+ annotate("text",x=c(1,2,3,4),y=c(5.5,4.4,4,5.5),size=14,label=c("a","b","c","a"),fontface=2)+#statistical significance annotate("text",x=c(2.5),y=c(6),size=16,label=c("***"),fontface=2)+ labs(tag="a")+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=35), axis.text.y=element_text(colour="black",size=35), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(4,"cm"), legend.key=element_blank(), legend.position="right") print(fig.1a)</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="acidification-of-formicine-ant-crop-lumens-through-swallowing-of-acidic-poison-gland-secretions"> Acidification of formicine ant crop lumens through swallowing of acidic poison gland secretions.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">(<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>) The pH of crop lumens at 4 hr, 24 hr, and 48 hr after feeding <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants 10% honey water (pH = 5) at 0 hr and at 4 hr after re-feeding ants at 48 hr (LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.acidification.c.floridanus,m.acidification.c.floridanus0)[2,6],2)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.acidification.c.floridanus,m.acidification.c.floridanus0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.acidification.c.floridanus,m.acidification.c.floridanus0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.acidification.c.floridanus.glht$test)$pvalues[3],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.acidification.c.floridanus.glht$test)$pvalues[2],3))</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). </p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1b" title="Figure 1b"> <label data-itemprop="label">Figure 1b</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 50 #' @height 30 #############plot fig.1b #calculation of median values and confidence intervals of predictor ant manipulation ph.summary.mobility.c.floridanus<-ddply(mobility.c.floridanus,"treatment",summarize, N=length(ph), median=quantile(ph,0.5), lowerCI=quantile(ph,0.025), upperCI=quantile(ph,0.975)) #addition of median values per ant manipulation into the data.frame as a new variable mobility.c.floridanus2<-mobility.c.floridanus%>%group_by(treatment)%>%mutate(medianph=median(ph)) #definition of universal ph indicator colors: 1-6 ph.colors.rgb1<-c( "202 58 67", "235 81 42", "252 125 19", "253 190 2", "254 250 0", "235 230 54" ) #transformation of RGB colors into hex colors ph.colors.hex1<-sapply(strsplit(ph.colors.rgb1, " "), function(ph.colors.rgb1) rgb(ph.colors.rgb1[1], ph.colors.rgb1[2], ph.colors.rgb1[3], maxColorValue=255)) #plotting of Fig.1b fig.1b<-ggplot()+ geom_boxplot(data=mobility.c.floridanus2,mapping=aes(x=treatment,y=ph,fill=medianph,colour=treatment),outlier.shape=NA,lwd=1.5,alpha=0.5,width=0.6,coef=0)+ scale_fill_gradientn(colours=ph.colors.hex1,breaks=c(1,2,3,4,5,6),limits=c(1,6.5),guide=FALSE)+ scale_color_manual(values=c("grey70","grey30"),guide=FALSE)+ geom_point(data = mobility.c.floridanus, mapping=aes(x=treatment,y=ph,fill=ph),pch=21,color="white",position=position_jitter(width=0.1),size=6.5,alpha=1)+ scale_x_discrete(name="Treatment",labels=c("FA -","FA +"))+ scale_y_continuous(name="pH-value",limits=c(1,6),breaks=c(1,2,3,4,5,6))+ geom_segment(aes(x = 1, y = 5.5, xend = 2, yend = 5.5),lwd=1.5)+ annotate("text",x=c(1.5),y=c(5.6),size=16,label=c("***"),fontface=2)+#statistical significance labs(tag="b")+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=35), axis.text.y=element_text(colour="black",size=35), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(4,"cm"), legend.key=element_blank(), legend.position="right") fig.1b</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="acidification-of-formicine-ant-crop-lumens-through-swallowing-of-acidic-poison-gland-secretions-1"> Acidification of formicine ant crop lumens through swallowing of acidic poison gland secretions.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">(<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>) The pH of crop lumens in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants that were either prevented to ingest the formic acid containing poison gland secretion (FA-) or not (FA+) for 24 hr after feeding (LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround( anova(m.mobility.c.floridanus,m.mobility.c.floridanus0)[2,6],2)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.mobility.c.floridanus,m.mobility.c.floridanus0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( format_pval(myround(anova(m.mobility.c.floridanus,m.mobility.c.floridanus0)[2,8],3)))</code><output slot="output"></output></stencila-code-expression>). </p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1c" title="Figure 1c"> <label data-itemprop="label">Figure 1c</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 80 #' @height 50 #############plot fig.1c mobility.formicine.ants$species2<-factor(mobility.formicine.ants$species,levels=c("maculatus","fuliginosus","cinerea","cunicularia","fuscocinerea","pratensis","rufibarbis"))#reorder facotr species #calculation of median values and confidence intervals of predictor ant manipulation (two levels:FA- and FA+) and of factor species (six levels) ph.summary.mobility.formicine.ants<-ddply(mobility.formicine.ants,c("species2","treatment"),summarize, N=length(ph), median=quantile(ph,0.5), lowerCI=quantile(ph,0.025), upperCI=quantile(ph,0.975)) #addition of median values per ant manipulation into the data.frame as a new variable mobility.formicine.ants2<-mobility.formicine.ants%>%group_by(colony,treatment)%>%mutate(medianph=median(ph)) #definition of universal ph indicator colors: 1-6 ph.colors.rgb1<-c( "202 58 67", "235 81 42", "252 125 19", "253 190 2", "254 250 0", "235 230 54" ) #transformation of RGB colors into hex colors ph.colors.hex1<-sapply(strsplit(ph.colors.rgb1, " "), function(ph.colors.rgb1) rgb(ph.colors.rgb1[1], ph.colors.rgb1[2], ph.colors.rgb1[3], maxColorValue=255)) #plotting of Fig.1c fig.1c<-ggplot()+ geom_boxplot(data=mobility.formicine.ants2,mapping=aes(x=species2,y=ph,fill=medianph,colour=treatment),position=position_dodge(0.5),outlier.shape=NA,lwd=1.5,alpha=0.5,width=0.4,coef=0)+ scale_fill_gradientn(colours=ph.colors.hex1,guide=FALSE,breaks=c(1,2,3,4,5,6),limits=c(1,6.5))+ scale_color_manual(values=c("grey70","grey30"),name="Animal treatment:",labels=c("FA-","FA+"))+ geom_point(data = mobility.formicine.ants, mapping=aes(x=species2,y=ph,fill=ph,group=treatment),pch=21,color="white",position=position_jitterdodge(jitter.width=0.1,jitter.height=0.05,dodge.width=0.5),size=5,alpha=1)+ scale_x_discrete(name="Species", labels=c( expression(paste(italic("C. maculatus"))), expression(paste(italic("L. fuliginosus"))), expression(paste(italic("F. cinerea"))), expression(paste(italic("F.cunicularia"))), expression(paste(italic("F. fuscocinerea"))), expression(paste(italic("F. pratensis"))), expression(paste(italic("F. rufibarbis"))) ))+ scale_y_continuous(name="pH-value",limits=c(1,6),breaks=c(1,2,3,4,5,6))+ geom_segment(aes(x = 0.875, y = 3.9, xend = 1.125, yend = 3.9),lwd=2)+ geom_segment(aes(x = 1.875, y = 3.9, xend = 2.125, yend = 3.9),lwd=2)+ geom_segment(aes(x = 2.875, y = 4.9, xend = 3.125, yend = 4.9),lwd=2)+ geom_segment(aes(x = 3.875, y = 4.9, xend = 4.125, yend = 4.9),lwd=2)+ geom_segment(aes(x = 4.875, y = 4.9, xend = 5.125, yend = 4.9),lwd=2)+ geom_segment(aes(x = 5.875, y = 5.4, xend = 6.125, yend = 5.4),lwd=2)+ geom_segment(aes(x = 6.875, y = 4.4, xend = 7.125, yend = 4.4),lwd=2)+ annotate("text",x=c(1,2,3,4,5,6,7),y=c(4.1,4.1,5.1,5.1,5.1,5.6,4.6),size=8,label= c( expression(paste(italic("P")," = 0.036")), expression(paste(italic("P")," < 0.001")), expression(paste(italic("P")," < 0.001")), expression(paste(italic("P")," = 0.007")), expression(paste(italic("P")," < 0.001")), expression(paste(italic("P")," = 0.018")), expression(paste(italic("P")," < 0.001"))), fontface=1)+ labs(tag="c")+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=30,angle=50,hjust=1), axis.text.y=element_text(colour="black",size=30), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=30), legend.text=element_text(size=30), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(4,"cm"), legend.key=element_blank(), legend.position="right") fig.1c</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="acidification-of-formicine-ant-crop-lumens-through-swallowing-of-acidic-poison-gland-secretions-2"> Acidification of formicine ant crop lumens through swallowing of acidic poison gland secretions.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">(<strong itemscope="" itemtype="http://schema.stenci.la/Strong">c</strong>) The pH-value of crop lumens 24 hr after feeding in seven formicine ant species that were either prevented to ingest the formic acid containing poison gland secretion (FA-) or not (FA+). Wilcoxon rank-sum tests (two-sided). Lines and shaded boxes show the median and interquartile range; points show all data. Colors in shaded rectangles near the y-axis represent universal indicator pH colors. Color filling of shaded boxes correspond to the median pH color of x-axis groups and color filling of points correspond to universal indicator pH colors. The border of shaded boxes represents animal treatment (light gray: prevention of poison ingestion, FA-; dark gray: poison ingestion not prevented, FA+).</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s1" title="Figure 1—figure supplement 1."><label data-itemprop="label">Figure 1—figure supplement 1.</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #addition of median values per status and caste into the data.frame as a new variable acidification.baseline2<-acidification.baseline%>%group_by(status,caste)%>%mutate(medianph=median(ph)) #definition of universal ph indicator colors: 1-6 ph.colors.rgb1<-c( "202 58 67", "235 81 42", "252 125 19", "253 190 2", "254 250 0", "235 230 54" ) #transformation of RGB colors into hex colors ph.colors.hex1<-sapply(strsplit(ph.colors.rgb1, " "), function(ph.colors.rgb1) rgb(ph.colors.rgb1[1], ph.colors.rgb1[2], ph.colors.rgb1[3], maxColorValue=255)) #plotting of Fig.1 - figure supplement 1 fig.1.figure.supplement1<-ggplot()+ geom_boxplot(data=acidification.baseline2,mapping=aes(x=status,y=ph,fill=medianph,colour=caste),position=position_dodge(0.5),outlier.shape=NA,lwd=1.5,alpha=0.5,width=0.4,coef=0)+ scale_fill_gradientn(colours=ph.colors.hex1,guide=FALSE,breaks=c(1,2,3,4,5,6),limits=c(1,6.5))+ scale_color_manual(values=c("grey70","grey30"),name="Worker caste:",labels=c("Major","Minor"))+ geom_point(data = acidification.baseline, mapping=aes(x=status,y=ph,fill=ph,group=caste),pch=21,color="white",position=position_jitterdodge(jitter.width=0.1,jitter.height=0.05,dodge.width=0.5),size=5,alpha=1)+ scale_x_discrete(name="Animal feeding status",labels=c("Colony:\nsatiated","Colony cohort:\nstarved"))+ scale_y_continuous(name="pH-value",limits=c(1,6),breaks=c(1,2,3,4,5,6))+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=30,angle=30,hjust=1), axis.text.y=element_text(colour="black",size=30), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=30), legend.text=element_text(size=30), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(4,"cm"), legend.key=element_blank(), legend.position="right") fig.1.figure.supplement1</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="baseline-acidity-of-c-floridanus-crop-lumens-under-satiated-and-starved-conditions"> Baseline acidity of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> crop lumens under satiated and starved conditions.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">pH of crop lumens in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> workers (light gray: major workers, dark gray: minor workers) that were either taken directly out of a satiated colony or that were kept in cohorts of ~100 individuals under satiated conditions for 3 d and then starved for 24 hr before measuring the pH. Lines and shaded boxes show the median and interquartile range; points show all data. Colors in shaded rectangles near the y-axis represent universal indicator pH colors. Color filling of shaded boxes correspond to the median pH color of x-axis groups and color filling of points correspond to universal indicator pH colors. Border of shaded boxes represents animal caste (light gray: major workers; dark gray: minor workers).</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s2" title="Figure 1—figure supplement 2."><label data-itemprop="label">Figure 1—figure supplement 2.</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 ############plot Fig. 1 - figure supplement 2 fig.1.figure.supplement2<-ggplot()+ geom_boxplot(data=acidopore.grooming,mapping=aes(x=food.treatment1,y=groomingevents,fill=food.treatment1),outlier.shape=NA,lwd=1.5,alpha=0.2,width=0.4,coef=0)+ geom_point(data=acidopore.grooming,mapping=aes(x=food.treatment1,y=groomingevents,color=food.treatment1),position=position_jitter(width=0.1),size=4,alpha=0.7)+ scale_fill_manual(values=c("white","white","white"),name="Food treatment (Mean/Median):",labels=c("unfed","water","honey"),guide=FALSE)+ scale_colour_manual(values=c("grey","dodgerblue","yellow1"),name="Raw data",guide=FALSE)+ scale_x_discrete(name="Food",labels=c("unfed","water","honey"))+ scale_y_continuous(name="Acidopre grooming frequency",limits=c(-0.5,15))+ annotate("text",x=c(1,2,3),y=c(8,11,11),size=10,label=c("a","b","b"),fontface=2)+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=18), axis.text.y=element_text(colour="black",size=18), axis.title.x=element_text(colour="black",size=25,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=25,margin=margin(0,22,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), legend.position="right") fig.1.figure.supplement2 </code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="acidopore-grooming-frequency-of-c-floridanus-after-ingestion-of-different-food-types"> Acidopore grooming frequency of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> after ingestion of different food types.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Frequency of acidopore grooming within 30 min. after fluid ingestion (water or 10% honey water) compared to ants that did not receive any fluid (unfed) (GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.grooming,m.grooming0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.grooming,m.grooming0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.grooming,m.grooming0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>,same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.grooming.glht$test)$pvalues[2],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.grooming.glht$test)$pvalues[1],3))</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons).</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s3" title="Figure 1—figure supplement 3."><label data-itemprop="label">Figure 1—figure supplement 3.</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig.1 - figure supplement 3 #addition of median values per position into the data.frame as a new variable ph.midgut2<-ph.midgut%>%group_by(gutregion)%>%mutate(medianph=median(ph,na.rm=TRUE)) #definition of universal ph indicator colors: 1-7 ph.colors.rgb<-c( "202 58 67", "235 81 42", "252 125 19", "253 190 2", "254 250 0", "235 230 54", "133 203 40" ) #transformation of RGB colors into hex colors ph.colors.hex<-sapply(strsplit(ph.colors.rgb, " "), function(ph.colors.rgb) rgb(ph.colors.rgb[1], ph.colors.rgb[2], ph.colors.rgb[3], maxColorValue=255)) #plotting of Fig.1 - figure supplement 3 fig.1.figure.supplement3<-ggplot()+ geom_boxplot(data=ph.midgut2,mapping=aes(x=gutregion,y=ph,fill=medianph),colour="grey30",lwd=1.5,outlier.shape=NA,alpha=0.5,coef=0)+ scale_fill_gradientn(colours=ph.colors.hex,guide=FALSE,breaks=c(1,2,3,4,5,6,7),limits=c(1,7.5))+ geom_point(data=ph.midgut2,mapping=aes(x=gutregion,y=ph,fill=ph),pch=21,color="white",position=position_jitter(width=0.1),size=6.5,alpha=1)+ scale_x_discrete(name="Gut position",labels=c("Crop","Midgut\nposition 1","Midgut\nposition 2","Midgut\nposition 3","Midgut\nposition 4"))+ scale_y_continuous(name="pH-value",limits=c(1,7),breaks=c(1,2,3,4,5,6,7))+ annotate("text",x=c(1,2,3,4,5),y=c(4.5,6.5,6.5,6.5,6.5),size=9,label=c("a","b","b","b","b"),fontface=2)+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=20), axis.text.y=element_text(colour="black",size=20), axis.title.x=element_text(colour="black",size=25,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=25,margin=margin(0,22,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(3.6,"cm"), legend.key=element_blank(), legend.position="right") fig.1.figure.supplement3</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="acidity-along-the-gastrointestinal-tract-of-c-floridanus">Acidity along the gastrointestinal tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em>.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">pH-measurements 24 hr after access to 10% honey-water in the crop (N = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.midgut[1,2]</code><output slot="output"></output> </stencila-code-expression>) and directly after the proventriculus at four points along the midgut (N = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.midgut[2,2]</code><output slot="output"></output> </stencila-code-expression> except position four with N = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">ph.summary.midgut[5,2]</code><output slot="output"></output> </stencila-code-expression>) (LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.ph.midgut,m.ph.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.ph.midgut,m.ph.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.ph.midgut,m.ph.midgut0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p≥ <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.ph.midgut.glht$test)$pvalues[6],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.ph.midgut.glht$test)$pvalues[1],3))</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). Lines and shaded boxes show the median and interquartile range; points show all data. Colors in shaded rectangles near the y-axis represent universal indicator pH colors. Color filling of shaded boxes correspond to the median pH color of x-axis groups and color filling of points correspond to universal indicator pH colors.</p> </figcaption> </figure> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To pinpoint acidopore grooming and swallowing of the poison gland secretion as the source for crop acidity and to exclude that internal, physiological mechanisms cause crop acidity, we then prevented acidopore grooming in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants for 24 hr after feeding through immobilization. This experiment revealed that acidopore grooming prevented ants showed a significantly diminished acidity in their crop compared to ants that were not prevented from acidopore grooming (<a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1b</a>; LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.mobility.c.floridanus,m.mobility.c.floridanus0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.mobility.c.floridanus,m.mobility.c.floridanus0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.mobility.c.floridanus,m.mobility.c.floridanus0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>). A similar, significantly diminished acidity in crop lumens was ubiquitously obtained in a comparative survey across seven formicine ant species and three genera (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus</em>, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Lasius</em>, and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica</em>) upon comparison of ants that were prevented from acidopore grooming through immobilization to non-prevented ants (<a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1c</a>; two-sided Wilcoxon rank-sum tests, comparisons for all ant species: p≤ <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(wilcox.test(ph~treatment,data=mobility.c.maculatus)$p.value,3)</code><output slot="output"></output></stencila-code-expression>). We conclude that formicine ants attain a highly acidic baseline pH in their crop lumen by taking up their poison into their mouth during acidopore grooming (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib140">bib140</a></cite>), and subsequently swallowing it. The comparative survey also shows that this behavior is widespread among formicine ants.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Although venomous animals often bear a cost of venom production and express behavioural adaptations to limit venom expenditure (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib27">bib27</a></cite>), we also found that <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants increase the frequency of acidopore grooming within the first 30 min after ingesting fluids compared to unfed ants irrespective of the fluid’s nutritional value (<a href="#fig1s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 2</a>; GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.grooming,m.grooming0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.grooming,m.grooming0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.grooming,m.grooming0)[2,8],3))</code><output slot="output"></output> </stencila-code-expression>; Westfall corrected post-hoc pairwise comparisons, water versus. 10% honey-water: p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.grooming.glht$test)$pvalues[2],3)</code><output slot="output"></output></stencila-code-expression>, unfed versus water and unfed versus 10% honey-water: both p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.grooming.glht$test)$pvalues[1],3))</code><output slot="output"></output></stencila-code-expression>). Moreover, we found that the strong acidity was limited to the crop of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants and did not extend to the midgut, the primary site of digestion in insects (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib66">bib66</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib137">bib137</a></cite>; <a href="#fig1s3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 3</a>; pH-measurements at four points along the midgut 24 hr after access to 10% honey-water; mean ± se; midgut position 1 = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(ph.summary.midgut[2,6],2)</code><output slot="output"></output> </stencila-code-expression> ± <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(ph.summary.midgut[2,9],2)</code><output slot="output"></output> </stencila-code-expression>, midgut position 2 = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.midgut[3,6],2)</code><output slot="output"></output></stencila-code-expression> ± <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.midgut[3,9],2)</code><output slot="output"></output></stencila-code-expression>, midgut position 3 = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(ph.summary.midgut[4,6],2)</code><output slot="output"></output> </stencila-code-expression> ± <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(ph.summary.midgut[4,9],2)</code><output slot="output"></output> </stencila-code-expression>, midgut position 4 = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.midgut[5,6],2)</code><output slot="output"></output></stencila-code-expression> ± <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(ph.summary.midgut[5,9],2)</code><output slot="output"></output></stencila-code-expression>). Together, these results led us to hypothesize that poison acidified crop lumens in formicine ants do not primarily serve a digestive function but may serve microbial control, limiting infection by oral pathogens. </p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="poison-acidified-crops-can-prevent-the-passage-of-pathogenic-and-opportunistic-bacteria-to-the-midgut"> Poison acidified crops can prevent the passage of pathogenic and opportunistic bacteria to the midgut</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To investigate a potential microbial control function, we next tested whether poison acidified crop lumens can inhibit <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em>, an insect pathogenic bacterium (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib54">bib54</a></cite>), when ingested together with food and prevent its passage from the crop to the midgut in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants. To this end, we first estimated food passage times through the gut of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> with fluorescent particles contained in food, as we surmised that ingested fluids need to remain in the crop for a minimum time before being passed to the midgut in order for poison swallowing and the ensuing crop acidity to take effect after perturbation of the crop pH through ingested fluids. In agreement with food passage times through the gastrointestinal tract of other ants (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib25">bib25</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib79">bib79</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib78">bib78</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib67">bib67</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib91">bib91</a></cite>), we found that only a small amount of ingested food is passed from the crop to the midgut 2–4 hr after feeding, while thereafter food is steadily passed from the crop to the midgut until 18 hr after feeding (<a href="#fig2s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 1</a>). </p> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>###################################################################################################### ############################### Fig 2 a,b: Serratia viability in vivo ################################ ###################################################################################################### #############loading data for Fig.2a,b growth.serratia<-read.delim("https://datadryad.org/stash/downloads/file_stream/452663",header=T)#choose data file: Supplementary Data Fig.2a,b.txt #str(growth.serratia) #create factor variable time2 from the continuous variable time growth.serratia$time2<-as.factor(growth.serratia$time) #subsetting the data frame into two groups (serratia.crop, serratia.midgut) growth.serratia.crop<-subset(growth.serratia,gutregion=="crop") growth.serratia.crop<-drop.levels(growth.serratia.crop) growth.serratia.midgut<-subset(growth.serratia,gutregion=="midgut") growth.serratia.midgut<-drop.levels(growth.serratia.midgut) #calculation of median values and confidence intervals of change in CFUs relative to 0h in the crop growth.serratia.summary<-ddply(growth.serratia,c("gutregion","time"),summarise, N=length(cfu.change.relative), median=quantile(cfu.change.relative,0.5), lowerCI=quantile(cfu.change.relative,0.025), upperCI=quantile(cfu.change.relative,0.975)) #growth.serratia.summary #removal of time factor levels 4h and 48h in the midgut of serratia fed ants, as these levels consisted only of zeros and were not included in the statistical analysis growth.serratia.midgut1<-subset(growth.serratia.midgut,time2!="48"&time2!="4") growth.serratia.midgut1<-drop.levels(growth.serratia.midgut1) #############statistical analysis Fig. 2a m.growth.serratia.crop<-glmmTMB(cfu~time2+(1|colony),ziformula=~1,data=growth.serratia.crop,family=nbinom1) #summary(m.growth.serratia.crop) #model validation res.m.growth.serratia.crop<-simulateResiduals(fittedModel=m.growth.serratia.crop) #plot(res.m.growth.serratia.crop) #significance of predictor time since feeding (five levels: 0h, 0.5h, 4h, 24h, 48h) m.growth.serratia.crop0<-glmmTMB(cfu~1+(1|colony),ziformula=~1,data=growth.serratia.crop,family=nbinom1) #anova(m.growth.serratia.crop,m.growth.serratia.crop0) #post-hoc comporisons of predictor-levels time since feeding m.growth.serratia.crop.glht<-glht_glmmTMB(m.growth.serratia.crop,linfct=mcp(time2="Tukey")) output.m.growth.serratia.crop.glht<-summary(m.growth.serratia.crop.glht,test=adjusted("Westfall")) #output.m.growth.serratia.crop.glht #############statistical analysis Fig. 2b m.growth.serratia.midgut<-glmmTMB(cfu~time2+(1|colony),data=growth.serratia.midgut1,ziformula=~1,family=nbinom2) #summary(m.growth.serratia.midgut) #model validation res.m.growth.serratia.midgut<-simulateResiduals(fittedModel=m.growth.serratia.midgut) #plot(res.m.growth.serratia.midgut) #statistical significance of predictor time (three levels: 0h, 0.5h, 24h) m.growth.serratia.midgut0<-glmmTMB(cfu~1+(1|colony),data=growth.serratia.midgut1,ziformula=~1,family=nbinom2) #anova(m.growth.serratia.midgut,m.growth.serratia.midgut0) ###################################################################################################### ############ Fig. 2 - figure supplement 1a,b: food passage fluorescent particles ##################### ###################################################################################################### #############loading data for Fig. 2 - figure supplement 1a,b food.flow<-read.delim("https://datadryad.org/stash/downloads/file_stream/452664",header=T)#choose data file: Supplementary Data Fig.2 - figure supplement 1a,b.txt #str(food.flow) food.flow$time<-factor(food.flow$time,levels=c("2h","4h","6h","8h","12h","14h","16h","18h","24h","48h"))#reorder factor time food.flow$gutregion<-factor(food.flow$gutregion,levels=c("crop","midgut","hindgut"))#reorder factor gutregion #calculation of median values and confidence intervals of particles in gutregion over time per minor and major workers food.flow.summary<-ddply(food.flow,c("caste","gutregion","time"),summarise, N=length(particles), median=quantile(particles,0.5), lowerCI=quantile(particles,0.025), upperCI=quantile(particles,0.975)) #food.flow.summary #subsetting the data frame food.flow into two groups (food.flow.minor, food.flow.major) food.flow.minor<-subset(food.flow,caste=="minor") food.flow.minor<-drop.levels(food.flow.minor) food.flow.major<-subset(food.flow,caste=="major") food.flow.major<-drop.levels(food.flow.major) ###################################################################################################### ################### Fig. 2 - figure supplement 2a,b: E.coli viability in vivo ######################## ###################################################################################################### #############loading data for Fig. 2 - figure supplement 2a,b growth.ecoli<-read.delim("https://datadryad.org/stash/downloads/file_stream/452665",header=T)#choose data file: Supplementary Data Fig.2 - figure supplement 2a,b.txt #str(growth.ecoli) #create factor variable time2 from the continuous variable time growth.ecoli$time2<-as.factor(growth.ecoli$time) #subsetting the data frame into two groups (ecoli.crop, ecoli.midgut) growth.ecoli.crop<-subset(growth.ecoli,gutregion=="crop") growth.ecoli.crop<-drop.levels(growth.ecoli.crop) growth.ecoli.midgut<-subset(growth.ecoli,gutregion=="midgut") growth.ecoli.midgut<-drop.levels(growth.ecoli.midgut) #calculation of median values and confidence intervals of change in CFUs relative to 0h in the crop growth.ecoli.summary<-ddply(growth.ecoli,c("gutregion","time"),summarise, N=length(cfu.change.relative), median=quantile(cfu.change.relative,0.5), lowerCI=quantile(cfu.change.relative,0.025), upperCI=quantile(cfu.change.relative,0.975)) #growth.ecoli.summary #removal of time factor levels 0h in the midgut of e.coli fed ants, as this level consisted only of zeros and were not included in the statistical analysis growth.ecoli.midgut1<-subset(growth.ecoli.midgut,time2!="0") growth.ecoli.midgut1<-drop.levels(growth.ecoli.midgut1) #############statistical analysis Fig. 2 - figure supplement 2a m.growth.ecoli.crop<-glmmTMB(cfu~time2+colony,data=growth.ecoli.crop,ziformula=~1,family=nbinom1) #summary(m.growth.ecoli.crop) #model validation res.m.growth.ecoli.crop<-simulateResiduals(fittedModel=m.growth.ecoli.crop) #plot(res.m.growth.ecoli.crop) #statistical significance of predictor time since feeding (five levels: 0h, 0.5h, 4h, 24h, 48h) m.growth.ecoli.crop0<-glmmTMB(cfu~colony,data=growth.ecoli.crop,ziformula=~1,family=nbinom1) #anova(m.growth.ecoli.crop,m.growth.ecoli.crop0) #statistical significance of predictor colony (four levels: C313, C322, C333, C94) m.growth.ecoli.crop1<-glmmTMB(cfu~time2,data=growth.ecoli.crop,ziformula=~1,family=nbinom1) #anova(m.growth.ecoli.crop,m.growth.ecoli.crop1) #post-hoc comporisons of predictor-levels time since feeding m.growth.ecoli.crop1.glht<-glht_glmmTMB(m.growth.ecoli.crop1,linfct=mcp(time2="Tukey")) output.m.growth.ecoli.crop1.glht<-summary(m.growth.ecoli.crop1.glht,test=adjusted("Westfall")) #output.m.growth.ecoli.crop1.glht #############statistical analysis Fig. 2 - figure supplement 2b m.growth.ecoli.midgut<-glmmTMB(cfu~time2+(1|colony),data=growth.ecoli.midgut1,ziformula=~1,family=nbinom1) #summary(m.growth.ecoli.midgut) #model validation res.m.growth.ecoli.midgut<-simulateResiduals(fittedModel=m.growth.ecoli.midgut) #plot(res.m.growth.ecoli.midgut) #statistical significance of predictor time since feeding (four levels: 0.5h, 4h, 24h, 48h) m.growth.ecoli.midgut0<-glmmTMB(cfu~1+(1|colony),data=growth.ecoli.midgut1,ziformula=~1,family=nbinom1) #anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0) #post-hoc comporisons of predictor-levels time since feeding m.growth.ecoli.midgut.glht<-glht_glmmTMB(m.growth.ecoli.midgut,linfct=mcp(time2="Tukey")) output.m.growth.ecoli.midgut.glht<-summary(m.growth.ecoli.midgut.glht,test=adjusted("Westfall")) #output.m.growth.ecoli.midgut.glht ###################################################################################################### ##################### Fig. 2 - figure supplement 3: Serratia growth in vitro ######################### ###################################################################################################### #############loading data for Fig. 2 - figure supplement 3 serratia.growth.invitro<-read.delim("https://datadryad.org/stash/downloads/file_stream/452666",header=T)#choose file: Supplementary Data Fig. 2 - figure supplement 3.txt #str(serratia.growth.invitro) #calculation of median values and confidence intervals of change in CFUs relative to pH 5 serratia.growth.invitro.summary<-ddply(serratia.growth.invitro,c("ph"),summarize, median.cfu.change=quantile(cfu.change.relative,0.5), lowerCI.cfu.change=quantile(cfu.change.relative,0.025), upperCI.cfu.change=quantile(cfu.change.relative,0.975)) #serratia.growth.invitro.summary #removal of pH levels 2 and 3 from serratia.growth.invitro, as these levels consisted only of zeros serratia.growth.invitro1<-subset(serratia.growth.invitro,ph=="pH5"|ph=="pH4") serratia.growth.invitro1<-drop.levels(serratia.growth.invitro1) #str(serratia.growth.invitro1) #############statistical analysis Fig. 2 - figure supplement 3 m.serratia.growth.invitro1<-glm.nb(cfu~ph,data=serratia.growth.invitro1) #summary(m.serratia.growth.invitro1) #model validation res.m.serratia.growth.invitro1<-simulateResiduals(fittedModel=m.serratia.growth.invitro1) #plot(res.m.serratia.growth.invitro1) #statistical significance of predictor pH-level (two levels: pH5, pH4) m.serratia.growth.invitro10<-glm.nb(cfu~1,data=serratia.growth.invitro1) #anova(m.serratia.growth.invitro1,m.serratia.growth.invitro10)</code></pre> </stencila-code-chunk> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">We then measured the viability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em> ingested together with food in the gastrointestinal tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> at two time points before (0.5 hr and 4 hr) and after (24 hr and 48 hr) main food passage from the crop to the midgut, with the time directly after food ingestion (0 hr) serving as a reference. We found that <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> presence decreased sharply over time in the crop (<a href="#fig2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2a</a>; GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.serratia.crop,m.growth.serratia.crop0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.growth.serratia.crop,m.growth.serratia.crop0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.growth.serratia.crop,m.growth.serratia.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>). The proportion of CFUs that we were able to retrieve from the crop relative to the mean at 0 hr in the crop diminished from <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[2,4]*100)</code><output slot="output"></output></stencila-code-expression>% at 0.5 hr post-feeding (median, CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[2,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[2,6]*100)</code><output slot="output"></output></stencila-code-expression>%) to <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[3,4]*100)</code><output slot="output"></output></stencila-code-expression>% at 4 hr (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[3,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[3,6]*100)</code><output slot="output"></output></stencila-code-expression>%), 24 hr (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[4,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[4,6]*100)</code><output slot="output"></output></stencila-code-expression>%), and 48 hr (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[5,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[5,6]*100)</code><output slot="output"></output></stencila-code-expression>%) post-feeding. In addition, relative to the mean at 0 hr in the crop, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> could only be detected at extremely low numbers in the midgut (median <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[6,4]*100)</code><output slot="output"></output></stencila-code-expression>%) at 0 hr (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[6,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[6,6]*100)</code><output slot="output"></output></stencila-code-expression>%), 0.5 hr (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[7,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[7,6]*100)</code><output slot="output"></output></stencila-code-expression>%) and 24 hr (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.serratia.summary[9,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.serratia.summary[9,6]*100)</code><output slot="output"></output></stencila-code-expression>%) post-feeding and not at all at 4 hr and 48 hr post-feeding (<a href="#fig2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2b</a>; GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.serratia.midgut,m.growth.serratia.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.growth.serratia.midgut,m.growth.serratia.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.serratia.midgut,m.growth.serratia.midgut0)[2,8],3)</code><output slot="output"></output></stencila-code-expression>)). A similar, rapid reduction in the crop and inability to pass from the crop to the midgut was obtained when we fed <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli,</em> a potential opportunistic bacterium that is not a gut associate of insects (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib10">bib10</a></cite>) to <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants (<a href="#fig2s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 2</a>; crop: GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>; midgut: GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0)[2,8],3)</code><output slot="output"></output></stencila-code-expression>).</p> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig2" title="Figure 2."> <label data-itemprop="label">Figure 2.</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 2a fig.2a<-ggplot()+ geom_boxplot(data=growth.serratia.crop,aes(x=time2,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,alpha=0.3,coef=0,width=0.4)+ geom_point(data=growth.serratia.crop,aes(x=time2,y=cfu.change.relative),pch=21,fill="deeppink1",color="white",alpha=1,position=position_jitter(width=0.1),size=5)+ annotate("text",x=c(1,2,3,4,5),y=c(6.5,6.5,0.7,0.7,0.7),size=9,label=c("a","a","b","b","b"),fontface=2)+ scale_x_discrete(name="Time",labels=c("0h","0.5h","4h","24h","48h"))+ scale_y_continuous(name="CFU change",limits=c(-0.01,7.5))+ labs(tag="a")+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15,face="italic"), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), aspect.ratio=1) fig.2a fig.2b<-ggplot()+ geom_boxplot(data=growth.serratia.midgut,aes(x=time2,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,alpha=0.7,coef=0,width=0.4)+ geom_point(data=growth.serratia.midgut,aes(x=time2,y=cfu.change.relative),fill="deeppink1",pch=21, color="white",alpha=1,position=position_jitter(width=0.1),size=5)+ annotate("text",x=c(3),y=c(0.15),size=9,label=c(expression(paste(italic("P")," = 0.593"))),fontface=2)+ scale_x_discrete(name="Time",labels=c("0h","0.5h","4h","24h","48h"))+ scale_y_continuous(name="CFU change",limits=c(-0.01,0.17))+ labs(tag="b")+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15,face="italic"), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), aspect.ratio=1) fig.2b</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="viability-of-s-marcescens-over-time-in-the-digestive-tract-of-c-floridanus"> Viability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> over time in the digestive tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em>.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Change in the number of colony forming units (CFUs) in the crop (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>) and midgut (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>) part of the digestive tract (yellow color in insert) relative to the mean CFU-number at 0 hr in the crop (CFU change corresponds to single data CFU-value divided by mean CFU-value at 0 hr in the crop), 0 hr, 0.5 hr, 4 hr, 24 hr, and 48 hr after feeding <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em> ants 10% honey water contaminated with <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em>. (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>), Change of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> in the crop (GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.serratia.crop,m.growth.serratia.crop0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.growth.serratia.crop,m.growth.serratia.crop0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.growth.serratia.crop,m.growth.serratia.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p≥ <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.growth.serratia.crop.glht$test)$pvalues[8],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.growth.serratia.crop.glht$test)$pvalues[2],3))</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>), Change of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> in the midgut (GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.serratia.midgut,m.growth.serratia.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.growth.serratia.midgut,m.growth.serratia.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.serratia.midgut,m.growth.serratia.midgut0)[2,8],3)</code><output slot="output"></output></stencila-code-expression>). Note that timepoints with zero bacterial growth in the midgut (4 hr and 48 hr) were excluded from the statistical model.</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig2s1" title="Figure 2—figure supplement 1"><label data-itemprop="label">Figure 2—figure supplement 1</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig.2 - figure supplement 1a #subsetting the data frame food.flow.summary into three groups (food.flow.summary.minor.crop, food.flow.summary.minor.midgut, food.flow.summary.minor.hindgut) food.flow.summary.minor<-subset(food.flow.summary,caste=="minor") food.flow.summary.minor<-drop.levels(food.flow.summary.minor) food.flow.summary.minor.crop<-subset(food.flow.summary.minor,gutregion=="crop") food.flow.summary.minor.crop<-drop.levels(food.flow.summary.minor.crop) food.flow.summary.minor.midgut<-subset(food.flow.summary.minor,gutregion=="midgut") food.flow.summary.minor.midgut<-drop.levels(food.flow.summary.minor.midgut) food.flow.summary.minor.hindgut<-subset(food.flow.summary.minor,gutregion=="hindgut") food.flow.summary.minor.hindgut<-drop.levels(food.flow.summary.minor.hindgut) #plot Fig.2 - figure supplement 1a fig.2.figure.supplement1a<-ggplot()+ geom_line(data = food.flow.summary.minor.crop,aes(x=time,y=median+1,group="time"),colour="blue",lwd=1.5,alpha=0.8)+ geom_line(data = food.flow.summary.minor.midgut,aes(x=time,y=median+1,group="time"),colour="skyblue",lwd=1.5,alpha=0.8)+ geom_line(data = food.flow.summary.minor.hindgut,aes(x=time,y=median+1,group="time"),colour="turquoise",lwd=1.5,alpha=0.8)+ geom_point(data = food.flow.minor,mapping=aes(x=time,y=particles+1,fill=gutregion),pch=21,color="white",position=position_jitter(width=0.1),size=7,alpha=1)+ scale_fill_manual(values=c("blue","skyblue","turquoise"))+ scale_x_discrete(name="Time after feeding",labels=c("2h","4h","6h","8h","12h","14h","16h","18h","24h","48h"))+ scale_y_continuous(trans="log10",name="Number particles",limits=c(-1,3000))+ annotation_logticks(sides="l",size=1, short = unit(0.25, "cm"), mid = unit(0.5, "cm"), long = unit(0.8, "cm"))+ labs(tag="a")+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=35), axis.text.y=element_text(colour="black",size=35), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(4,"cm"), legend.key=element_blank(), legend.position="right") fig.2.figure.supplement1a #############plot Fig.2 - figure supplement 1b #subsetting the data frame food.flow.summary into three groups (food.flow.summary.major.crop, food.flow.summary.major.midgut, food.flow.summary.major.hindgut) food.flow.summary.major<-subset(food.flow.summary,caste=="major") food.flow.summary.major<-drop.levels(food.flow.summary.major) food.flow.summary.major.crop<-subset(food.flow.summary.major,gutregion=="crop") food.flow.summary.major.crop<-drop.levels(food.flow.summary.major.crop) food.flow.summary.major.midgut<-subset(food.flow.summary.major,gutregion=="midgut") food.flow.summary.major.midgut<-drop.levels(food.flow.summary.major.midgut) food.flow.summary.major.hindgut<-subset(food.flow.summary.major,gutregion=="hindgut") food.flow.summary.major.hindgut<-drop.levels(food.flow.summary.major.hindgut) #plot Fig.2 - figure supplement 1b fig.2.figure.supplement1b<-ggplot()+ geom_line(data = food.flow.summary.major.crop,aes(x=time,y=median+1,group="time"),colour="blue",lwd=1.5,alpha=0.8)+ geom_line(data = food.flow.summary.major.midgut,aes(x=time,y=median+1,group="time"),colour="skyblue",lwd=1.5,alpha=0.8)+ geom_line(data = food.flow.summary.major.hindgut,aes(x=time,y=median+1,group="time"),colour="turquoise",lwd=1.5,alpha=0.8)+ geom_point(data = food.flow.major,mapping=aes(x=time,y=particles+1,fill=gutregion),pch=21,color="white",position=position_jitter(width=0.1),size=7,alpha=1)+ scale_fill_manual(values=c("blue","skyblue","turquoise"))+ scale_x_discrete(name="Time after feeding",labels=c("2h","4h","6h","8h","12h","14h","16h","18h","24h","48h"))+ scale_y_continuous(trans="log10",name="Number particles",limits=c(-1,3000))+ annotation_logticks(sides="l",size=1, short = unit(0.25, "cm"), mid = unit(0.5, "cm"), long = unit(0.8, "cm"))+ labs(tag="b")+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=35), axis.text.y=element_text(colour="black",size=35), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key.height=unit(4,"cm"), legend.key=element_blank(), legend.position="right") fig.2.figure.supplement1b</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="food-passage-of-fluorescent-particles-through-the-digestive-tract-of-c-floridanus"> Food passage of fluorescent particles through the digestive tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em>.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Number of fluorescent particles on a logarithmic scale in the crop (dark blue), midgut (light blue), and hindgut (turquoise) part of the digestive tract of minor (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>) and major (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>) ants 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 14 hr, 16 hr, 18 hr, 24 hr, and 48 hr after feeding them a 1:1 honey-water mix with polymethylmethacrylate (PMM) particles (size ≤40 µm). Note that for displaying purposes and better visibility of zero values a value of one has been added to all datapoints. Points represent the number of counted particles per individual ant and lines connect the median value of particles at the different time points after feeding.</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig2s2" title="Figure 2—figure supplement 2"><label data-itemprop="label">Figure 2—figure supplement 2</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 2 - figure supplement 2a fig.2.figure.supplement2a<-ggplot()+ geom_boxplot(data=growth.ecoli.crop,aes(x=time2,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,alpha=0.3,coef=0,width=0.4)+ geom_point(data=growth.ecoli.crop,aes(x=time2,y=cfu.change.relative),pch=21,fill="darkolivegreen",color="white",alpha=1,size=5,position=position_jitter(width=0.1))+ annotate("text",x=c(1,2,3,4,5),y=c(2.5,1.5,1,0.5,0.5),size=9,label=c("a","b","c","d","d"),fontface=2)+#statistical significance scale_x_discrete(name="Time",labels=c("0h","0.5h","4h","24h","48h"))+ scale_y_continuous(name="CFU change",limits=c(-0.01,3))+ labs(tag="a")+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15,face="italic"), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), aspect.ratio=1) fig.2.figure.supplement2a #############plot Fig. 2 - figure supplement 2b fig.2.figure.supplement2b<-ggplot()+ geom_boxplot(data=growth.ecoli.midgut,aes(x=time2,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,alpha=0.3,coef=0,width=0.4)+ geom_point(data=growth.ecoli.midgut,aes(x=time2,y=cfu.change.relative),pch=21,fill="darkolivegreen",color="white",alpha=1,size=5,position=position_jitter(width=0.1))+ annotate("text",x=c(2,3,4,5),y=c(0.02,0.02,0.02,0.02),size=9,label=c("ab","a","a","b"),fontface=2)+ scale_x_discrete(name="Time",labels=c("0h","0.5h","4h","24h","48h"))+ scale_y_continuous(name="CFU change",limits=c(-0.01,0.05))+ labs(tag="b")+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15,face="italic"), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), aspect.ratio=1) fig.2.figure.supplement2b </code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="viability-of-e-coli-over-time-in-the-digestive-tract-of-c-floridanus-over-time"> Viability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> over time in the digestive tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> over time.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Change in the number of colony forming units (CFUs) in the crop (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>) and midgut (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>) part of the digestive tract (yellow color in insert) relative to mean CFU-number at 0 hr in the crop (CFU change corresponds to single data CFU-value divided by the mean CFU-value at 0 hr in the crop), 0 hr, 0.5 hr, 4 hr, 24 hr, and 48 hr after feeding ants 10% honey water contaminated with <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Escherichia coli</em>. (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>), Change of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> in the crop (GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.growth.ecoli.crop1.glht$test)$pvalues[10],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p<<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.growth.ecoli.crop1.glht$test)$pvalues[5],3)</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>), Change of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> in the midgut (GLMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.ecoli.midgut,m.growth.ecoli.midgut0)[2,8],3)</code><output slot="output"></output></stencila-code-expression>, same letters indicate p≥ <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.growth.ecoli.midgut.glht$test)$pvalues[3],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p≤<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.growth.ecoli.midgut.glht$test)$pvalues[1],3)</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). Note that timepoints with zero bacterial growth in the midgut (0 hr) were excluded from the statistical model.</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig2s3" title="Figure 2—figure supplement 3"><label data-itemprop="label">Figure 2—figure supplement 3</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 2 - figure supplement 3 fig.2.figure.supplement3<-ggplot()+ geom_boxplot(data=serratia.growth.invitro,aes(x=ph,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,position=position_dodge(0.6),alpha=0.6,coef=0,width=0.4)+ geom_point(data=serratia.growth.invitro,aes(x=ph,y=cfu.change.relative,colour=ph),position=position_jitterdodge(dodge=0.6),size=4,alpha=0.8)+ scale_colour_manual(values=c("deeppink1","deeppink1","deeppink1","deeppink1"),name="Raw data",guide=FALSE)+ scale_x_discrete(name="pH of honeywater",labels=c("2","3","4","5"))+ scale_y_continuous(name="CFU change",limits=c(0,1.7))+ annotate("text",x=c(3.5),y=c(1.5),size=16,label=c(expression(paste(italic("P")," < 0.001"))),fontface=2)+#statistical significance geom_segment(aes(x = 3, y = 1.4, xend = 4, yend = 1.4),lwd=2)+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=30), axis.text.y=element_text(colour="black",size=30), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), legend.position="bottom", aspect.ratio=1) fig.2.figure.supplement3</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="s-marcescens-growth-in-vitro"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> growth in vitro.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Change in the number of CFUs relative to the mean at pH 5 (CFU change corresponds to single data CFU-value divided by the mean CFU-value at pH 5) after incubation of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em> in 10% honey water (pH = 5) or in 10% honey water acidified with commercial formic acid to a pH of 4, 3, or 2 for 2 hr (GLM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.serratia.growth.invitro1,m.serratia.growth.invitro10)[2,7],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.serratia.growth.invitro1,m.serratia.growth.invitro10)[2,6]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>). Note that pH-values with zero bacterial growth (pH 2 and 3) were excluded from the statistical model.</p> </figcaption> </figure> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Although in vivo the antimicrobial activity of the natural poison is likely higher than the antimicrobial activity of formic acid, the main component of the formicine poison gland secretion (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib88">bib88</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib110">bib110</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib128">bib128</a></cite>) due to the presence of other components (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib140">bib140</a></cite>), we then tested the ability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> to withstand acidic conditions created with formic acid in an in vitro experiment. We found that incubation of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> for 2 hr in 10% honey water acidified with formic acid to pH 4 resulted in a significantly lower number of CFUs relative to pH 5 and in zero growth for incubations at pH-levels that were lower than 4 (<a href="#fig2s3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 3</a>; GLM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.serratia.growth.invitro1,m.serratia.growth.invitro10)[2,7],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.serratia.growth.invitro1,m.serratia.growth.invitro10)[2,6]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.growth.ecoli.crop,m.growth.ecoli.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>). Our data thus indicate that poison acidified crops can indeed serve microbial control in formicine ants, likely inhibiting bacteria according to their ability to cope with acidic environments (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib89">bib89</a></cite>).</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="access-to-the-poison-improves-survival-upon-ingestion-of-pathogen-contaminated-food"> Access to the poison improves survival upon ingestion of pathogen contaminated food</h3> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>###################################################################################################### ############################### Fig 3: Individual survival ########################################### ###################################################################################################### #############loading data for Fig.3 survival<-read.delim("https://datadryad.org/stash/downloads/file_stream/452667",header=T,stringsAsFactors=T)#choose data file: Supplementary Data Fig.3.txt #str(survival) #############statistcal analysis for Fig.3 m.survival<-coxme(Surv(time.death.hours,status)~treatment+(1|experiment.block)+(1|colony),data=survival) #print(m.survival) #significance of predictor ant treatment (four levels: Serratia- FA-, Serratia- FA+, Serratia-+FA-, Serratia+ FA+) m.survival0<-coxme(Surv(time.death.hours,status)~1+(1|experiment.block)+(1|colony),data=survival) #anova(m.survival,m.survival0) #post-hoc comporisons of predictor-levels ant treatment m.survival.glht<-glht(m.survival,linfct=mcp(treatment="Tukey")) output.m.survival.glht<-summary(m.survival.glht,test=adjusted("Westfall")) #output.m.survival.glht</code></pre> </stencila-code-chunk> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To test whether acidic crops also provide a fitness benefit upon ingestion of pathogen contaminated food, we prevented acidopore grooming through immobilization in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants for 24 hr after feeding them once with 5 µL of either <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> contaminated honey water or non-contaminated honey water and monitored their survival thereafter without providing additional food. We found that acidopore access after pathogen ingestion significantly increased the survival probability of ants (<a href="#fig3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3</a>; COXME, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.survival,m.survival0)[2,2],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.survival,m.survival0)[2,3]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.survival,m.survival0)[2,4],4)</code><output slot="output"></output></stencila-code-expression>). The survival of ants prevented from acidopore grooming and fed once with the pathogen contaminated food was significantly lower than that of non-prevented ants fed the same food source (Westfall corrected post-hoc comparisons: FA - | <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em> presence + versus. all other ant groups: p≤<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.survival.glht$test)$pvalues[6],3)</code><output slot="output"></output> </stencila-code-expression>). In contrast, non-prevented ants fed once with the pathogen contaminated food source did not differ in survival to prevented and non-prevented ants fed the non-contaminated food source (Westfall corrected post-hoc comparisons: FA + | <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em> presence + versus. FA + | <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em> presence – and FA + | <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em> presence + versus. FA + | <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em> presence –: p≥ <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.survival.glht$test)$pvalues[1],3)</code><output slot="output"></output> </stencila-code-expression> for both comparisons). Although we observed an overall high mortality in this experimental setup, likely due to starvation following the one time feeding in combination with social isolation of individually kept ants (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib81">bib81</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib83">bib83</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib135">bib135</a></cite>), this result indicates that poison acidified crop lumens provide a fitness benefit in terms of survival to formicine ants upon ingestion of pathogen contaminated food.</p> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig3" title="Figure 3"> <label data-itemprop="label">Figure 3</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 3 fig.3<-ggplot(survival,aes(time=time.death.hours,status=status,linetype=treatment,colour=treatment))+ geom_km(lwd=2)+ scale_colour_manual(values=c("grey","grey","black","black"),name="Animal treatment",label=c("Serratia- | FA-","Serratia- | FA+","Serratia+ | FA-","Serratia+ | FA+"))+ scale_linetype_manual(values=c("solid","dashed","solid","dashed"),name="Animal treatment",labels=c("Serratia- | FA-","Serratia- | FA+","Serratia+ | FA-","Serratia+ | FA+"))+ scale_x_continuous(name="Time after feeding [h]",limits=c(0,160),breaks=seq(0,120,24))+ scale_y_continuous(name="Proportion survival",limits=c(0,1))+ geom_segment(aes(x=140,y=0.12,xend=150,yend=0.12),lwd=1.5,col="grey",lty=2)+ geom_segment(aes(x=140,y=0.10,xend=150,yend=0.10),lwd=1.5,col="black",lty=2)+ geom_segment(aes(x=140,y=0.08,xend=150,yend=0.08),lwd=1.5,col="grey",lty=1)+ geom_segment(aes(x=140,y=0.00,xend=150,yend=0.00),lwd=1.5,col="black",lty=1)+ annotate("text",x=c(155,155),y=c(0.105,0.005),size=10,label=c("a","b"),fontface=2)+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.5,"cm"), legend.key=element_blank(), legend.position=c(0.8,0.8), aspect.ratio=1) fig.3</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="survival-after-ingestion-of-pathogen-contaminated-food">Survival after ingestion of pathogen contaminated food.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Survival of individual <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants that were either prevented to ingest the formic acid containing poison gland secretion (FA-; ant outlines with blue dot) or not (FA+) after feeding them once either honey water contaminated with <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em> (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em>+, yellow circle with pink dots and black ant outlines) or non-contaminated honey water (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em>-) without providing food thereafter (COXME, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.survival,m.survival0)[2,2],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.survival,m.survival0)[2,3]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.survival,m.survival0)[2,4],4)</code><output slot="output"></output></stencila-code-expression>, same letters indicate p≥ <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.survival.glht$test)$pvalues[1],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p≤<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(( output.m.survival.glht$test)$pvalues[6],3)</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons).</p> </figcaption> </figure> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="access-to-the-poison-in-donor-ants-also-benefits-receiver-ants-without-poison-access-after-food-exchange-via-trophallaxis"> Access to the poison in donor ants also benefits receiver ants without poison access after food exchange via trophallaxis</h3> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>###################################################################################################### #################################### Fig 4: Survival trophallaxis #################################### ###################################################################################################### #############loading data for Fig.4 survival.trophallaxis<-read.delim("https://datadryad.org/stash/downloads/file_stream/452668",header=T,stringsAsFactors=T)#choose data file: Supplementary Data Fig.4.txt #str(survival) #############statistical analysis for Fig. 4 m.survival.trophallaxis<-coxme(Surv(time.death,status.death)~treatment+(1|colony)+(1|experiment.block)+(1|petridish),data=survival.trophallaxis) #print(m.survival.trophallaxis) #significance of predictor ant treatment (four levels: donor FA+, donor FA-, receiver FA+, receiver FA-) m.survival.trophallaxis0<-coxme(Surv(time.death,status.death)~1+(1|colony)+(1|experiment.block)+(1|petridish),data=survival.trophallaxis) #anova(m.survival.trophallaxis,m.survival.trophallaxis0) #post-hoc comparisons of predictor-levels ant treatment m.survival.trophallaxis.glht<-glht(m.survival.trophallaxis,linfct=mcp(treatment="Tukey")) output.m.survival.trophallaxis.glht<-summary(m.survival.trophallaxis.glht,test=adjusted("Westfall")) #output.m.survival.trophallaxis.glht #hazard ratios between predictor-levels ant treatment through releveling of predictor treatment1<-factor(survival.trophallaxis$treatment,levels=c("receiverFA+","receiverFA-","donorFA+","donorFA-")) treatment2<-factor(survival.trophallaxis$treatment,levels=c("donorFA+","receiverFA+","receiverFA-","donorFA-")) treatment3<-factor(survival.trophallaxis$treatment,levels=c("receiverFA-","receiverFA+","donorFA+","donorFA-")) m.survival.trophallaxis1<-coxme(Surv(time.death,status.death)~treatment1+(1|colony)+(1|experiment.block)+(1|petridish),data=survival.trophallaxis) #print(m.survival.trophallaxis1) m.survival.trophallaxis2<-coxme(Surv(time.death,status.death)~treatment2+(1|colony)+(1|experiment.block)+(1|petridish),data=survival.trophallaxis) #print(m.survival.trophallaxis2) m.survival.trophallaxis3<-coxme(Surv(time.death,status.death)~treatment3+(1|colony)+(1|experiment.block)+(1|petridish),data=survival.trophallaxis) #print(m.survival.trophallaxis3) ##################################################################################################### ######################### Fig.4 - figure supplement 1: trophallaxis behavior ######################## ##################################################################################################### #############loading data for Fig.4 - figure supplement 1 trophallaxis<-read.delim("https://datadryad.org/stash/downloads/file_stream/452669",header=T,stringsAsFactors=T)#choose file: Supplementary Data Fig.4 - figure supplement 1.txt #str(trophallaxis) #############statistcal analysis for Fig.4 - figure supplement 1 m.duration.trophallaxis<-lmer(sqrt(sum.duration.trophallaxis)~ant.pair+(1|colony)+(1|experimental.block),data=trophallaxis) #summary(m.duration.trophallaxis) #model validation res.m.duration.trophallaxis<-simulateResiduals(fittedModel=m.duration.trophallaxis) #plot(res.m.duration.trophallaxis) #significance of predictor food treatment (three levels: unfed, water, honey) m.duration.trophallaxis0<-lmer(sqrt(sum.duration.trophallaxis)~1+(1|colony)+(1|experimental.block),data=trophallaxis) #anova(m.duration.trophallaxis,m.duration.trophallaxis0)</code></pre> </stencila-code-chunk> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The ability to swallow the acidic poison may not only improve survival of formicine ants feeding directly on pathogen contaminated food but also of ants that share the contaminated food via trophallaxis. To test this, we created two types of donor-receiver ant pairs. Donor ants in both pairs were directly fed <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> contaminated food every other day, while receiver ants obtained food only through trophallaxis from their respective donor ants. Receiver ants in both pairs were precluded from swallowing of the poison through blockage of their acidopore opening, while donor ants were blocked in one pair but only sham blocked in the other pair. We found that the duration of trophallaxis between the two donor-receiver ant pairs during the first 30 min. of the first feeding bout did not significantly differ (<a href="#fig4s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 4—figure supplement 1</a>; LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.duration.trophallaxis,m.duration.trophallaxis0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.duration.trophallaxis,m.duration.trophallaxis0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.duration.trophallaxis,m.duration.trophallaxis0)[2,8],3)</code><output slot="output"></output></stencila-code-expression>), indicating that trophallactic behavior was not influenced through acidopore blockage in donor ants at the beginning of the experiment. Over the next 12 d, we found that acidopore blockage per se had a significant negative effect on the survival of donor as well as receiver ants (<a href="#fig4" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 4</a>; COXME, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.survival.trophallaxis,m.survival.trophallaxis0)[2,2],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.survival.trophallaxis,m.survival.trophallaxis0)[2,3]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.survival.trophallaxis,m.survival.trophallaxis0)[2,4],3))</code><output slot="output"></output></stencila-code-expression>). However, although receiver ants that obtained food every other day from donors with the ability to swallow the poison died at a higher rate than their respective donor counterparts (hazard ratio: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(exp(m.survival.trophallaxis2$coefficients)[1],2)</code><output slot="output"></output></stencila-code-expression>; Westfall corrected post-hoc comparison: p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.survival.trophallaxis.glht$test)$pvalues[5],3))</code><output slot="output"></output></stencila-code-expression>) they were only half as likely to die compared to receiver ants that obtained pathogen contaminated food from blocked donors unable to swallow their poison (hazard ratio: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(exp(m.survival.trophallaxis3$coefficients)[1],2)</code><output slot="output"></output></stencila-code-expression>; Westfall corrected post-hoc comparison: p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.survival.trophallaxis.glht$test)$pvalues[6],3))</code><output slot="output"></output></stencila-code-expression>). This indicates that swallowing of the poison and the ensuing crop acidity also provides a fitness benefit to other members of a formicine ant society.</p> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig4" title="Figure 4"> <label data-itemprop="label">Figure 4</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 4 fig.4<-ggplot(survival.trophallaxis,aes(time=time.death,,status=status.death,linetype=treatment,colour=treatment))+ geom_km(lwd=2)+ scale_colour_manual(values=c("grey","grey","black","black"),name="Animal:",labels=c("donor FA-","donor FA+","receiver FA- with\ndonor FA-","receiver FA- with\ndonor FA+"))+ scale_linetype_manual(values=c("dashed","solid","dashed","solid"),name="Animal:",label=c("donor FA-","donor FA+","receiver FA- with\ndonor FA-","receiver FA- with\ndonor FA+"))+ scale_x_continuous(name="Time in days",breaks=seq(0,14,2))+ scale_y_continuous(name="Proportion survival",limits=c(0,1))+ annotate("text",x=c(12.5,12.5,12.5,12.5),y=c(0.26,0.17,0.1,0.06),size=10,label=c("a","b","c","c"),fontface=2)+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.5,"cm"), legend.key=element_blank(), legend.position=c(0.8,0.8), aspect.ratio=1) fig.4</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="survival-after-sharing-pathogen-contaminated-food-via-trophallaxis">Survival after sharing pathogen contaminated food via trophallaxis.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Survival of donor ants (light gray ant outlines) that were directly fed with pathogen contaminated food (yellow circle with pink dots in insert) every other day and were either prevented to ingest their formic acid containing poison gland secretion (FA-; ant outlines with blue dot) or not (FA+) and survival of receiver ants (black ant outlines) that received pathogen contaminated food only through trophallaxis with donor ants and were always prevented to ingest their formic acid containing poison gland secretion (FA-) (COXME, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.survival.trophallaxis,m.survival.trophallaxis0)[2,2],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.survival.trophallaxis,m.survival.trophallaxis0)[2,3]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.survival.trophallaxis,m.survival.trophallaxis0)[2,4],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.survival.trophallaxis.glht$test)$pvalues[2],3)</code><output slot="output"></output></stencila-code-expression>) and different letters indicate p≤<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.survival.trophallaxis.glht$test)$pvalues[3],3)</code><output slot="output"></output></stencila-code-expression>) in Westfall corrected post hoc comparisons).</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig4s1" title="Figure 4—figure supplement 1"><label data-itemprop="label">Figure 4—figure supplement 1</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 fig.4.figure.supplement1<-ggplot()+ geom_boxplot(data=trophallaxis,mapping=aes(x=ant.pair,y=sum.duration.trophallaxis,fill=ant.pair),outlier.shape=NA,lwd=1.5,width=0.4,coef=0)+ geom_point(data=trophallaxis,mapping=aes(x=ant.pair,y=sum.duration.trophallaxis,color=ant.pair),position=position_jitter(width=0.1),size=4,alpha=0.9)+ scale_fill_manual(values=c("white","white"),name="Animal pairing:",labels=c("donorFA- receiverFA-","donorFA+ receiverFA-"),guide=FALSE)+ scale_colour_manual(values=c("deeppink1","deeppink1"),name="Raw data:",labels=c("no","yes"),guide=FALSE)+ scale_x_discrete(name="Animal pairing",labels=c("donorFA- receiverFA-","donorFA+ receiverFA-"))+ scale_y_continuous(name="Duration trophallaxis [s]",limits=c(-5,1800),breaks=seq(0,1800,200))+ geom_segment(aes(x = 1, y = 1750, xend = 2, yend = 1750),lwd=2)+ annotate("text",x=c(1.5),y=c(1800),size=10,label=c(expression(paste(italic("P")," = 0.268"))),fontface=2)+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=20), axis.text.y=element_text(colour="black",size=20), axis.title.x=element_text(colour="black",size=25,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=25,margin=margin(0,22,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), legend.position="right") fig.4.figure.supplement1</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="duration-of-trophallaxis-in-donor-receiver-ant-pairs">Duration of trophallaxis in donor-receiver ant pairs.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Total duration of trophallaxis events within 30 min. of the first bout of food exchange between donor-receiver ant-pairs (LMM, LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.duration.trophallaxis,m.duration.trophallaxis0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.duration.trophallaxis,m.duration.trophallaxis0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.duration.trophallaxis,m.duration.trophallaxis0)[2,8],3)</code><output slot="output"></output></stencila-code-expression>). Donor ants in both pairs were directly fed with <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em> contaminated 10% honey water and were either prevented to ingest their formic acid containing poison gland secretion (FA-) or not (FA+), while receiver ants received pathogen contaminated food only through trophallaxis with the respective donor ants and were always prevented to ingest their formic acid containing poison gland secretion (FA-). </p> </figcaption> </figure> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="poison-acidified-crops-allow-members-of-the-bacteria-family-acetobacteraceae-passage-to-the-midgut"> Poison acidified crops allow members of the bacteria family Acetobacteraceae passage to the midgut</h3> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>###################################################################################################### ################################ Fig 5 a,b: Asaia viability in vivo ################################## ###################################################################################################### #############loading data for Fig.5a,b growth.asaia<-read.delim("https://datadryad.org/stash/downloads/file_stream/452670",header=T,stringsAsFactors=T)#choose data file: Supplementary Data Fig.5a,b.txt #str(growth.asaia) #create factor variable time2 from the continuous variable time growth.asaia$time2<-as.factor(growth.asaia$time) #subsetting the data frame into two groups (growth.asaia.crop, growth.asaia.midgut) growth.asaia.crop<-subset(growth.asaia,gutregion=="crop") growth.asaia.crop<-drop.levels(growth.asaia.crop) growth.asaia.midgut<-subset(growth.asaia,gutregion=="midgut") growth.asaia.midgut<-drop.levels(growth.asaia.midgut) #calculation of median values and confidence intervals of change in CFUs relative to 0h in the crop growth.asaia.summary<-ddply(growth.asaia,c("gutregion","time"),summarise, N=length(cfu.change.relative), median=quantile(cfu.change.relative,0.5), lowerCI=quantile(cfu.change.relative,0.025), upperCI=quantile(cfu.change.relative,0.975)) #growth.asaia.summary #removal of time factor levels 0h in the midgut of asaia fed ants, as this level consisted only of zeros and is not included in the statistical analysis growth.asaia.midgut1<-subset(growth.asaia.midgut,time2!="0") growth.asaia.midugt1<-drop.levels(growth.asaia.midgut1) #############statistical analysis Fig. 5a m.growth.asaia.crop<-glmmTMB(cfu~time2+(1|colony),data=growth.asaia.crop,ziformula=~1,family=nbinom2) #summary(m.growth.asaia.crop) #model validation res.m.growth.asaia.crop<-simulateResiduals(fittedModel=m.growth.asaia.crop) #plot(res.m.growth.asaia.crop) #statistical significance of predictor time since feeding (five levels: 0h, 0.5h, 4h, 24h, 48h) m.growth.asaia.crop0<-glmmTMB(cfu~1+(1|colony),data=growth.asaia.crop,ziformula=~1,family=nbinom2) #anova(m.growth.asaia.crop,m.growth.asaia.crop0) #post-hoc comporisons of predictor-levels time since feeding m.growth.asaia.crop.glht<-glht_glmmTMB(m.growth.asaia.crop,linfct=mcp(time2="Tukey")) output.m.growth.asaia.crop.glht<-summary(m.growth.asaia.crop.glht,test=adjusted("Westfall")) #output.m.growth.asaia.crop.glht #############statistical analysis Fig. 5b m.growth.asaia.midgut<-glmmTMB(cfu~time2+(1|colony),data=growth.asaia.midgut1,ziformula=~1,family=nbinom1) #summary(m.growth.asaia.midgut) #model validation res.m.growth.asaia.midgut<-simulateResiduals(fittedModel=m.growth.asaia.midgut) #plot(res.m.growth.asaia.midgut) #statistical significance of predictor time since feeding (four levels: 0.5h, 4h, 24h, 48h) m.growth.asaia.midgut0<-glmmTMB(cfu~1+(1|colony),data=growth.asaia.midgut1,ziformula=~1,family=nbinom1) #anova(m.growth.asaia.midgut,m.growth.asaia.midgut0) #post-hoc comporisons of predictor-levels time since feeding m.growth.asaia.midgut.glht<-glht_glmmTMB(m.growth.asaia.midgut,linfct=mcp(time2="Tukey")) output.m.growth.asaia.midgut.glht<-summary(m.growth.asaia.midgut.glht,test=adjusted("Westfall")) #output.m.growth.asaia.midgut.glht ###################################################################################################### ##################### Fig. 5 - figure supplement 1: Asaia growth in vitro ############################ ###################################################################################################### #############loading data for Fig. 5 - figure supplement 1 asaia.growth.invitro<-read.delim("https://datadryad.org/stash/downloads/file_stream/452671",header=T,stringsAsFactors=T)#choose file: Supplementary Data Fig. 5 - figure supplement 1.txt #str(asaia.growth.invitro) #calculation of median values and confidence intervals of change in CFUs relative to pH 5 asaia.growth.invitro.summary<-ddply(asaia.growth.invitro,c("ph"),summarize, median.cfu.change=quantile(cfu.change.relative,0.5), lowerCI.cfu.change=quantile(cfu.change.relative,0.025), upperCI.cfu.change=quantile(cfu.change.relative,0.975)) #asaia.growth.invitro.summary #removal of pH level 2 from asaia.growth.invitro, as this level consisted only of zeros asaia.growth.invitro1<-subset(asaia.growth.invitro,ph!="pH2") asaia.growth.invitro1<-drop.levels(asaia.growth.invitro1) #str(asaia.growth.invitro1) #############statistical analysis Fig. 5 - figure supplement 1 m.asaia.growth.invitro1<-glm.nb(cfu~ph,data=asaia.growth.invitro1) #summary(m.asaia.growth.invitro1) #model validation res.m.asaia.growth.invitro1<-simulateResiduals(fittedModel=m.asaia.growth.invitro1) #plot(res.m.asaia.growth.invitro1) #statistical significance of predictor pH-level (two levels: pH5, pH4) m.asaia.growth.invitro10<-glm.nb(cfu~1,data=asaia.growth.invitro1) #anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10) #post-hoc comparison among predictor levels of pH (three levels: pH5, pH4, pH3) m.asaia.growth.invitro1.glht<-glht(m.asaia.growth.invitro1,linfct=mcp(ph="Tukey")) output.m.asaia.growth.invitro1.glht<-summary(m.asaia.growth.invitro1.glht,test=adjusted("Westfall")) #output.m.asaia.growth.invitro1.glht</code></pre> </stencila-code-chunk> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In addition to microbial control, poison acidified formicine ant crops might act as a chemical filter for gut-associated microbial communities, similar to gut morphological structures that can act as mechanical filters in ants (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib25">bib25</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib51">bib51</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib85">bib85</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib87">bib87</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib117">bib117</a></cite>) and other insects (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib71">bib71</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib107">bib107</a></cite>). To investigate the idea of a chemical filter, we tested the ability of the insect gut-associated bacterium <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. (family Acetobacteraceae) (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib34">bib34</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib44">bib44</a></cite>) to withstand acidic environments in vitro and in vivo. In contrast to <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> (<a href="#fig2s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 2</a>), <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. was not affected by an incubation for 2 hr in 10% honey water acidified with formic acid to pH 4 and was still able to grow when incubated at pH 3 in in vitro tests (<a href="#fig5s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5—figure supplement 1</a>; GLM, overall LR-test χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10)[2,7],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10)[2,6]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval( myround(anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10)[2,8],3))</code><output slot="output"></output></stencila-code-expression>; Westfall corrected post hoc comparisons: pH = 5 versus. pH = 4: p=<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.asaia.growth.invitro1.glht$test)$pvalues[3],3)</code><output slot="output"></output></stencila-code-expression>, all other comparisons: p <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.asaia.growth.invitro1.glht$test)$pvalues[1],3))</code><output slot="output"></output></stencila-code-expression>). Moreover, in in vivo tests, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. only gradually diminished over time in the crop (<a href="#fig5" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5a</a>; GLMM; LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.asaia.crop,m.growth.asaia.crop0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.growth.asaia.crop,m.growth.asaia.crop0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval(myround(anova(m.growth.asaia.crop,m.growth.asaia.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>) with the proportion of CFUs that we were able to retrieve from the crop relative to the mean at 0 hr in the crop diminishing to only <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.asaia.summary[3,4]*100)</code><output slot="output"></output> </stencila-code-expression>% (median, CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.asaia.summary[3,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.asaia.summary[3,6]*100)</code><output slot="output"></output></stencila-code-expression>%) and <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.asaia.summary[4,4]*100)</code><output slot="output"></output></stencila-code-expression>% (CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.asaia.summary[4,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.asaia.summary[4,6]*100)</code><output slot="output"></output></stencila-code-expression>%) at 4 hr and 24 hr post-feeding, respectively. At the same time, relative to the mean at 0 hr in the crop, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. steadily increased in the midgut (<a href="#fig5" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5b</a>; GLMM; LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">myround(anova(m.growth.asaia.midgut,m.growth.asaia.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">anova(m.growth.asaia.midgut,m.growth.asaia.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">format_pval(myround(anova(m.growth.asaia.midgut,m.growth.asaia.midgut0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>) from its initial absence at 0 hr post-feeding to <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.asaia.summary[10,4]*100)</code><output slot="output"></output></stencila-code-expression>% (median, CI: <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">round(growth.asaia.summary[10,5]*100)</code><output slot="output"></output></stencila-code-expression>–<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"> <code class="r" slot="text">round(growth.asaia.summary[10,6]*100)</code><output slot="output"></output></stencila-code-expression>%) at 48 hr post-feeding. This suggests that in formicine ants, poison acidified crops might act as a chemical filter that works selectively against the establishment of opportunistic and potentially harmful bacteria but allows entry and establishment of members of the bacterial family Acetobacteraceae.</p> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig5" title="Figure 5"> <label data-itemprop="label">Figure 5</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 5a fig.5a<-ggplot()+ geom_boxplot(data=growth.asaia.crop,aes(x=time2,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,alpha=0.3,coef=0,width=0.4)+ geom_point(data=growth.asaia.crop,aes(x=time2,y=cfu.change.relative),pch=21,fill="grey70",color="white",alpha=1,size=5,position=position_jitter(width=0.1))+ annotate("text",x=c(1,2,3,4,5),y=c(3,3,1.7,0.8,0.5),size=9,label=c("a","a","b","c","d"),fontface=2)+#statistical significance scale_x_discrete(name="Time",labels=c("0h","0.5h","4h","24h","48h"))+ scale_y_continuous(name="CFU change",limits=c(-0.01,4))+ labs(tag="a")+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15,face="italic"), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), aspect.ratio=1) fig.5a #############plot Fig. 5b fig.5b<-ggplot()+ geom_boxplot(data=growth.asaia.midgut,aes(x=time2,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,alpha=0.3,coef=0,width=0.4)+ geom_point(data=growth.asaia.midgut,aes(x=time2,y=cfu.change.relative),pch=21,fill="grey70",color="white",alpha=1,size=5,position=position_jitter(width=0.1))+ annotate("text",x=c(2,3,4,5),y=c(0.02,0.02,0.03,0.065),size=9,label=c("a","a","b","c"),fontface=2)+ scale_x_discrete(name="Time",labels=c("0h","0.5h","4h","24h","48h"))+ scale_y_continuous(name="Change in CFU's",limits=c(-0.01,0.065))+ labs(tag="b")+ theme(axis.line = element_line(colour = "black",size=2), axis.ticks=element_line(colour="black",size=2), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=25), axis.text.y=element_text(colour="black",size=25), axis.title.x=element_text(colour="black",size=30,margin=margin(25,0,0,0)), axis.title.y=element_text(colour="black",size=30,margin=margin(0,25,0,0)), plot.tag=element_text(colour="black",size=60,face="bold"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15,face="italic"), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), aspect.ratio=1) fig.5b </code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="viability-of-asaia-sp-over-time-in-the-digestive-tract-of-c-floridanus">Viability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia sp</em>. over time in the digestive tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em>.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Change in the number of colony forming units (CFUs) in the crop (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>) and midgut (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>) part of the digestive tract (yellow color in insert) relative to the mean CFU-number at 0 hr in the crop (CFU change corresponds to single data CFU-values divided by mean CFU-value at 0 hr in the crop), 0 hr, 0.5 hr, 4 hr, 24 hr, and 48 hr after feeding ants 10% honey water contaminated with <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">a</strong>), Change of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. in the crop (GLMM; LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.asaia.crop,m.growth.asaia.crop0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.growth.asaia.crop,m.growth.asaia.crop0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround(anova(m.growth.asaia.crop,m.growth.asaia.crop0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.growth.asaia.crop.glht$test)$pvalues[1],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p≤<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.growth.asaia.crop.glht$test)$pvalues[5],3)</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">b</strong>), Change of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. in the midgut (GLMM; LR-test, χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> =<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.growth.asaia.midgut,m.growth.asaia.midgut0)[2,6],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.growth.asaia.midgut,m.growth.asaia.midgut0)[2,7]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround(anova(m.growth.asaia.midgut,m.growth.asaia.midgut0)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.growth.asaia.midgut.glht$test)$pvalues[1],3)</code><output slot="output"></output></stencila-code-expression> and different letters indicate p≤<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.growth.asaia.midgut.glht$test)$pvalues[4],3)</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). Note that timepoints with zero bacterial growth in the midgut (0 hr) were excluded from the statistical model.</p> </figcaption> </figure> <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig5s1" title="Figure 5—figure supplement 1"><label data-itemprop="label">Figure 5—figure supplement 1</label> <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-programminglanguage="r"> <pre class="language-r" itemscope="" itemtype="http://schema.stenci.la/CodeBlock" slot="text"><code>#' @width 40 #' @height 30 #############plot Fig. 5 - figure supplement 1 fig.5.figure.supplement1<-ggplot()+ geom_boxplot(data=asaia.growth.invitro,aes(x=ph,y=cfu.change.relative),fill="white",outlier.shape=NA,lwd=1.5,position=position_dodge(0.6),alpha=0.6,coef=0,width=0.4)+ geom_point(data=asaia.growth.invitro,aes(x=ph,y=cfu.change.relative,colour=ph),position=position_jitterdodge(dodge=0.6),size=4,alpha=0.8)+ scale_colour_manual(values=c("grey40","grey40","grey40","grey40"),name="Raw data:",guide=FALSE)+ annotate("text",x=c(2,3,4),y=c(0.9,1.4,1.4),size=9,label=c("b","a","a"),fontface=2)+#statistical significance scale_x_discrete(name="pH of honeywater",labels=c("2","3","4","5"))+ scale_y_continuous(name="CFU change",limits=c(0,1.7))+ theme(axis.line = element_line(colour = "black",size=1.5), axis.ticks=element_line(colour="black",size=1.5), axis.ticks.length=unit(0.3,"cm"), axis.text.x=element_text(colour="black",size=30), axis.text.y=element_text(colour="black",size=30), axis.title.x=element_text(colour="black",size=40,margin=margin(20,0,0,0)), axis.title.y=element_text(colour="black",size=40,margin=margin(0,22,0,0)), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank(), legend.title=element_text(size=15), legend.text=element_text(size=15), legend.key.size=unit(1.2,"cm"), legend.key=element_blank(), legend.position="bottom", aspect.ratio=1) fig.5.figure.supplement1</code></pre> </stencila-code-chunk> <figcaption> <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="asaia-sp-growth-in-vitro"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia sp</em>. growth in vitro.</h4> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Change in the number of CFUs relative to the mean at pH 5 (CFU change corresponds to single data CFU-value divided by mean CFU-value at pH 5) after incubation of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. in 10% honey water (pH = 5) or in 10% honey water acidified with commercial formic acid to a pH of 4, 3, or 2 for 2 hr (GLM, LR-test χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround(anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10)[2,7],3)</code><output slot="output"></output></stencila-code-expression>, df = <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10)[2,6]</code><output slot="output"></output></stencila-code-expression>, p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval( myround(anova(m.asaia.growth.invitro1,m.asaia.growth.invitro10)[2,8],3))</code><output slot="output"></output></stencila-code-expression>, same letters indicate p= <stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">myround((output.m.asaia.growth.invitro1.glht$test)$pvalues[3],3)</code><output slot="output"></output></stencila-code-expression>, and different letters indicate p<stencila-code-expression programming-language="r" itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="r" slot="text">format_pval(myround((output.m.asaia.growth.invitro1.glht$test)$pvalues[1],3))</code><output slot="output"></output></stencila-code-expression> in Westfall corrected post hoc comparisons). Note that pH-values with zero bacterial growth (pH 2) were excluded from the statistical model.</p> </figcaption> </figure> <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="discussion">Discussion</h2> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In this study, we investigated how formicine ants solve the challenge to control harmful microbes in their food while at the same time allowing acquisition and transmission of beneficial microbes from and with their food. We found that formicine ants swallow their antimicrobial, highly acidic poison gland secretion during the behavior of acidopore grooming. The resulting acidic environment in their stomach, the crop, can protect formicine ants from food borne bacterial pathogens while at the same time allowing the acquisition and establishment of members of the bacterial family Acetobacteraceae, a recurring part of the gut microbiota of formicine ants.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Highly acidic stomach lumens are ubiquitous in higher vertebrates, including amphibians, reptiles, birds and mammals (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib7">bib7</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib80">bib80</a></cite>). In insects, highly acidic gut regions have so far only rarely been described from the midgut (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib28">bib28</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib66">bib66</a></cite>). The mechanisms responsible for the creation of a gut lumen compartment with a certain pH are often unknown in insects (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib57">bib57</a></cite>), but in principle, highly acidic gut regions in insects may, similar to vertebrates (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib60">bib60</a></cite>), be generated through physiological mechanisms (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib93">bib93</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib96">bib96</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib109">bib109</a></cite>). Alternatively, acidic derivatives of gut-associated microbes (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib122">bib122</a></cite>, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib123">bib123</a></cite>, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib149">bib149</a></cite>) or acidic gland secretions (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib11">bib11</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib100">bib100</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib143">bib143</a></cite>) might contribute to the insect gut pH. In agreement with the latter, the results of our study show that formicine ants maintain a highly acidic baseline pH in their stomach, the crop, through swallowing of their poison gland secretion during acidopore grooming. Interestingly, although we found that a higher crop acidity was observed in all formicine ants in our comparative survey when they had access to their poison, we also found that crop acidity was highly variable in ants with and without access to their poison. While a variable crop acidity in ants without access to their poison could indicate the existence of additional internal or external sources that maintain crop acidity, a variable crop acidity in ants with access to their poison could indicate species specific differences in acidopore grooming, in the composition of the poison gland secretion or in optimal crop acidity. Future studies will need to explore these possibilities.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Sanitation of food through the addition of organic acids or through acidic fermentation is frequently practiced by humans (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib29">bib29</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib64">bib64</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib139">bib139</a></cite>) and sanitation of food with antimicrobials from different sources is ubiquitous in animals that provision food to their offspring or that store, cultivate, develop or live in food (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib35">bib35</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib62">bib62</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib63">bib63</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib74">bib74</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib102">bib102</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib144">bib144</a></cite>). A microbial control function of poison acidified crops in formicine ants to sanitize ingested food is supported by our survival experiments and our in vivo and in vitro bacterial growth and viability experiments. There we found that access to the poison improved survival of formicine ants after feeding on pathogen contaminated food. We also found that pathogenic and opportunistic bacteria were quickly inhibited in the crop when ingested with food and could not establish in the midgut. Although our data suggests that this is likely due to the sensitivity of these bacteria to acidic environments, our evidence for this is only indirect. At present it is unclear whether the acidic environment in the crop is sufficient to protect formicine ants and to inhibit pathogenic and opportunistic microbes ingested with food or whether acidic conditions act in concert with other factors. Studies in vertebrates and the fruit fly <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Drosophila melanogaster</em> have shown that acidic gut regions together with immune system effectors serve microbial control and prevent infection by oral pathogens (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib50">bib50</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib68">bib68</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib92">bib92</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib113">bib113</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib119">bib119</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib133">bib133</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib136">bib136</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib147">bib147</a></cite>). Concordantly, previous studies investigating formicine ant trophallactic fluids <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib55">bib55</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib86">bib86</a></cite> found the presence of proteins related to cathepsin D, a lysosomal aspartic protease that can exhibit antibacterial effector activity and the proteolytic production of antimicrobial peptides (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib105">bib105</a></cite>). Future studies will therefore need to disentangle the relative contributions of crop acidity and immune system effectors released into the gut lumen to the improved survival of formicine ants in the face of pathogen contaminated food and to the microbe inhibitory action of poison acidified crops in formicine ants.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In addition to improving their own survival, the ability of donor ants to access their poison also improved the survival of receiver ants without access to their poison following trophallactic exchange of pathogen-contaminated food. Acidic crop lumens might therefore act as a barrier to disease spread in formicine ant societies, alleviating the cost of sharing pathogen contaminated food (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib108">bib108</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib125">bib125</a></cite>) and counteracting the generally increased risk of pathogen exposure and transmission associated with group-living (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib1">bib1</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib13">bib13</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib75">bib75</a></cite>). Although food distribution via trophallaxis is a dynamic process governed by many different factors (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib22">bib22</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib23">bib23</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib52">bib52</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib130">bib130</a></cite>), the technological advances in recent years to track multiple individuals of a group simultaneously over time (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib49">bib49</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib52">bib52</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib69">bib69</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib134">bib134</a></cite>), will make it possible to clarify the contribution of acidic crop lumens to disease spread prevention in formicine ant societies.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Acidic crop lumens might not only serve microbial control but might also act as a chemical filter for microbes, working selectively against pathogenic or opportunistic bacteria but allowing entry and establishment of species from the bacteria family Acetobacteraceae. We found that, compared to a bacterial pathogen, a bacterial member of the Acetobacteraceae was not only better able to withstand acidic conditions created with formic acid in vitro but was able to establish itself in the midgut of formicine ants in vivo. This suggests that host filtering of microbes (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib94">bib94</a></cite>) via acidic crop lumens might explain at least part of the recurrent presence of Acetobacteraceae in the gut of formicine ants and the otherwise reduced microbial diversity and abundance of gut-associated microbes in formicine ants (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib18">bib18</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib19">bib19</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib31">bib31</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib32">bib32</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib72">bib72</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib124">bib124</a></cite>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Though not formally established (see <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib103">bib103</a></cite>), recent studies indicate a mutualistic relationship between formicine ants, and their gut-associated Acetobacteraceae (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib19">bib19</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib32">bib32</a></cite>). Thus, the creation of an acidic crop environment in formicine ants that is easier to endure if colonizing microbes are mutualists agrees with the concept of screening as a mechanism to choose microbial partners out of a pool of environmental microbes (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib3">bib3</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib4">bib4</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib9">bib9</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib127">bib127</a></cite>). Contrary to signalling, where costly information is displayed to partners, in screening a costly environment is imposed on partners that excludes all but high-quality ones. Partner choice in a number of cross-kingdom mutualisms is readily explained by screening (see examples in <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib3">bib3</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib4">bib4</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib9">bib9</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib127">bib127</a></cite>) but experimental evidence is so far limited in insect-microbe associations (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib70">bib70</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib71">bib71</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib120">bib120</a></cite>). Although our experiments can only hint at screening as a means of partner choice in formicine ants, the results of our study would provide support for the prediction that screening is more likely to evolve from a host’s defense trait against parasites (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib3">bib3</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib4">bib4</a></cite>), that is, the highly acidic, antimicrobial poison that creates a selective environment for microbes. Our study might therefore not only provide evidence that the well-established cross talk between the immune system and gut-associated microbes in vertebrates and invertebrates (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib30">bib30</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib119">bib119</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib133">bib133</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib147">bib147</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib150">bib150</a></cite>) can hold for a broader range of immune defense traits (sensu <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib112">bib112</a></cite>) but also that this cross talk can be realized through signals (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib46">bib46</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib101">bib101</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib145">bib145</a></cite>) and through screening.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Overall, our study provides evidence that poison acidified crop lumens of formicine ants can act as a chemical filter for control and selection of microbes ingested with food. Poison acidified formicine crops might thus contribute to the ecological and evolutionary success of this group of insects by alleviating the increased risk of pathogen exposure and transmission associated with group living but allowing the acquisition and transmission of microbial mutualists. Similar microbial filters likely represent a widespread theme to manage harmful and beneficial host-associated microbes but have so far only partly been uncovered in a few animal systems (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib26">bib26</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib39">bib39</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib126">bib126</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib129">bib129</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib131">bib131</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib132">bib132</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib146">bib146</a></cite>).</p> <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="materials-and-methods"> Materials and methods</h2> <table id="keyresource" itemscope="" itemtype="http://schema.org/Table"> <caption><label data-itemprop="label">Key resources table</label></caption> <thead> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Reagent type (species) or resource</th> <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Designation</th> <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Source or reference</th> <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Identifiers</th> <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Additional information </th> </tr> </thead> <tbody> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus)</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus maculatus)</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus maculatus</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Lasius fuliginosus)</em> </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Lasius fuliginosus</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica cinerea)</em> </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica cinerea</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica cunicularia)</em> </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica cunicularia</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica fuscocinerea)</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica fuscocinerea</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica pratensis)</em> </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica pratensis</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Biological sample (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica rufibarbis)</em> </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica rufibarbis</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">See Materials and methods</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Strain, strain background (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens)</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Strain DSM12481, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Strain, strain background (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Escherichia coli)</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Escherichia coli</em></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Strain DSM6897, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Strain, strain background (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.)</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Strain SF2.1 <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib44">bib44</a></cite></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Blaubrand intraMARK micro pipettes</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Brand, Wertheim, Germany </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span data-itemtype="http://schema.org/Number">708707</span></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">pH sensitive paper</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Hartenstein, Würzburg, Germany</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">PHIP</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">pH electrode</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Unisense, Aarhus, Denmark</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Polymethylmethacrylate </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">University of Bayreuth, Animal Ecology I, group microplastic</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Leica microscope DM 2000 LED</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Leica, Wetzlar, Germany </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Leica stereomicroscope M 165 C</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Leica, Wetzlar, Germany </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Commercial honey</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Different brands</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">10% (w/v), 1:1 honey:water</td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Other</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">superglue</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">UHU brand</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Chemical compound, drug </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">≥95% Formic acid</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Sigmaaldrich, Merck, Darmstadt, Germany</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Cat# F0507</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Chemical compound, drug </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Trypton</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Sigmaaldrich, Merck, Darmstadt, Germany</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Cat# T7293-250G</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Chemical compound, drug </td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Yeast extract</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Millipore, Merck, Darmstadt, Germany</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Cat# Y1625-250G</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> </tr> <tr itemscope="" itemtype="http://schema.stenci.la/TableRow"> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Software, algorithm</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell">R version 3. 6.1</td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td> <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib118">bib118</a></cite></td> </tr> </tbody> </table> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="ant-species-and-maintenance">Ant species and maintenance</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Colonies of the carpenter ant <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em> were collected in 2001 and 2003 in Florida, USA, housed in Fluon (Whitford GmbH, Diez, Germany) coated plastic containers with plaster ground and maintained at a constant temperature of 25°C with 70% humidity and a 12 hr/12 hr light/dark cycle. They were given water ad libitum and were fed two times per week with honey water (1:1 tap water and commercial quality honey), cockroaches (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Blaptica dubia</em>) and an artificial diet (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib8">bib8</a></cite>). For comparison, workers of one other <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus</em> species (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus maculatus</em>), collected close to Kibale Forest, Uganda, in 2003 and housed under identical conditions as <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em> were used. Additionally, six other formicine ant species, one <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Lasius,</em> and five <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica</em> species (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Lasius fuliginosus, Formica cinerea, Formica cunicularia, Formica fuscocinerea, Formica pratensis,</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica rufibarbis</em>) were collected in Bayreuth, Germany in 2012 and 2018 and kept for approximately 2 weeks prior experimental use at 20°C, 70% humidity and a 14 hr/10 hr light/dark cycle. Except otherwise noted only the small worker caste (‘minors’) of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus</em> species was used.</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="acidity-of-the-crop-lumen-and-ph-measurements">Acidity of the crop lumen and pH measurements</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To determine whether formicine ants swallow their poison after feeding, we tracked changes in pH-levels of the crop lumen in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants over time. Before use in experimental settings, cohorts of ~100 ants were taken out of their natal colony (n = 6 colonies) into small plastic containers lined with Fluon and starved for 24–48 hr. Thereafter, ants were put singly into small petri dishes (Ø 55 mm) with damp filter paper covered bottom, given access to a droplet of 10% honey water (w/v) for 2 hr before removing the food source and measuring the pH of the crop lumen after another 2 hr (group 0+4 hr: n = 60 workers), after 24 hr (group 0+24 hr: n = 59 workers) or 48 hr (group 0+48 hr: n = 52 workers). To assess the effect of renewed feeding, a separate group of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants were given access to 10% honey water 48 hr after the first feeding for 2 hr prior to measuring the pH of their crop lumen after another 2 hr (group 48h+4 hr: n = 60 workers). To measure the pH, ants were first cold anesthetized on ice, then their gaster was cut off with a fine dissection scissor directly behind the petiole and leaking crop content (1–3 µL) collected with a capillary (5 µL Disposable Micro Pipettes, Blaubrand intraMARK, Brand, Wertheim). The collected crop content was then emptied on a pH sensitive paper to assess the pH (Hartenstein, Unitest pH 1–11). This method of collecting crop content will invariably result in some mixing of crop lumen content with haemolymph. As the pH of the insect haemolymph ranges from only slightly acidic (pH ≥6.5) to near-neutral or slightly alkaline (pH ≤8.2) (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib57">bib57</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib93">bib93</a></cite>), this might have biased the results of our pH measurements to slightly higher pH values. As a reference point for food pH, we also measured the pH of 10% honey water on pH sensitive paper, which gave invariably pH = 5. </p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In addition, we measured the pH in the crop lumen and at four points in the lumen along the midgut (1st measurement directly behind proventriculus to 4th measurement one mm apical from insertion point of the Malpighian tubules) of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> workers that were fed 24 hr prior to measurements with 10% honey-water. For these measurements worker guts were dissected as a whole and pH was measured in the crop (n = 2 workers from two colonies) and along the midgut (all midgut points n = 10, except point four with n = 9 workers from four different colonies) with a needle-shaped microelectrode (UNISENSE pH-meter; microelectrode with needle tip of 20 µm diameter).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In formicine ants, oral uptake of the poison into the mouth is performed via acidopore grooming (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib140">bib140</a></cite>). During this behavior ants bend their gaster forward between the legs and the head down to meet the acidopore, the opening of the poison gland, at the gaster tip (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib5">bib5</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib43">bib43</a></cite>). In an additional experiment we therefore compared the crop lumen pH of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> workers from four different colonies that were either prevented to reach their acidopore (FA- ants) or could reach their acidopore freely (FA+ ants). To do this, we again allowed single ants access to 10% honey water for 2 hr after a starvation period, before cold anesthetizing them briefly on ice and immobilizing FA- ants (n = 22 workers) in a pipetting tip, while FA+ ants (n = 23 workers) remained un-manipulated. After 24 hr we measured the pH of the crop lumen as before.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To investigate whether swallowing of the acidic poison is widespread among formicine ants, the latter experiment was repeated for six additional formicine ant species (FA- ants: n = 10 workers except for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica pratensis</em> with n = 21; FA+ ants: n = 10 workers except for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica pratensis</em> with n = 20; all ants: n = 1 colony) in the same fashion as described before with the exception that apart from <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica pratensis</em> the crop lumen was collected through the mouth by gently pressing the ants’ gaster. Crop lumen of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Formica pratensis</em> ants was collected in the same fashion as crop lumen of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To investigate whether the type of fluid and its nutritional value have an influence on the frequency of acidopore grooming in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em>, the following experiment was performed. Cohorts of ~100 ants were taken out of their natal colony (n = 6 colonies) into small plastic containers and starved for 24–48 hr. Thereafter, ants were again put singly into small petri dishes (Ø 55 mm) and given access to either a 3 µL droplet of 10% honey water (w/v, n = 126 ants, treatment: honey-water fed), a 3 µL droplet of tap water (n = 128, water-fed) or to no fluid (n = 125, unfed). After acclimatization (unfed ants) or after swallowing of the fluid (honey-water and water-fed ants, both 1–2 min.), all ants were filmed for the next 30 min. (Logitech webcam c910). These videos were then analyzed for the frequency of acidopore grooming.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Finally, we measured the pH in the crop lumen of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants (n = 3 colonies) under satiated and starved conditions to estimate a baseline level of acidity in the crop. For this, ants taken out of satiated, twice per week fed colonies on the day of feeding were compared to ants that were maintained in cohorts of ~100 individuals for 3 d with access to 10% honey-water and then starved for 24 hr before measuring the pH in their crop (n = 10 major and 10 minor workers per colony and condition). The pH in the crop lumen was measured as described before by briefly cold anesthetizing ants an ice, collecting the crop content through the mouth by gently pressing the ants’ gaster and then emptying it on a pH sensitive paper (Hartenstein, Unitest pH 1–11).</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="bacterial-strains-and-culture">Bacterial strains and culture</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">As model entomopathogenic bacterium <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia marcescens</em> DSM12481 (DSMZ Braunschweig, Germany) was used. This bacterium is pathogenic in a range of insects (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib54">bib54</a></cite>) and has been detected in formicine ants, that is <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Anoplolepis gracilipes</em> (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib33">bib33</a></cite>) and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Camponotus floridanus</em> (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib121">bib121</a></cite>). While often non-lethal within the digestive tract, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> can cross the insect gut wall (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib97">bib97</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib104">bib104</a></cite>) and is highly virulent upon entry into the hemocoel (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib47">bib47</a></cite>), not least due to the production of bacterial toxins (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib61">bib61</a></cite>). As a model bacterial gut-associate of ants <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. strain SF2.1 (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib44">bib44</a></cite>), was used. <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. belongs to the family Acetobacteraceae, members of which often thrive in sugar-rich environments (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib90">bib90</a></cite>), such as honeydew that ants like <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> predominantly feed on. <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. is capable of cross-colonizing insects of phylogenetically distant genera and orders (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib34">bib34</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib44">bib44</a></cite>) and can be a component of the gut-associated microbial community of formicine and other ants (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib31">bib31</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib76">bib76</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib77">bib77</a></cite>). In addition to <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia sp.</em>, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Escherichia coli</em> DSM6897 (DSMZ Braunschweig, Germany) was used as a model opportunistic bacterium that is not a gut-associate of insects. <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> bacteria are a principal constituent of mammalian gut-associated microbial communities but are commonly also found in the environment (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib10">bib10</a></cite>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Bacterial stocks of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens, Asaia</em> sp., and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> were kept in 25% glycerol at −80°C until use. For use, bacteria were plated on agar plates (LB-medium: 10 g tryptone, 5 g yeast extract, 20 g agar in 1L MilliQ-water, and GLY-medium: 25 g gycerol, 10 g yeast extract, 20 g agar in 1L MilliQ-water with pH adjusted to 5.0, for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens/E. coli</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. respectively), single colony forming units (CFUs) were picked after 24 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens/E. coli</em>) or 48 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.) of growth at 30°C and transferred to 5 ml liquid medium (LB-medium and GLY-medium minus agar for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens/E. coli</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. respectively) for an overnight culture (24 hr) at 30°C. The overnight culture was then pelleted by centrifugation at 3000 g, the medium discarded and resolved in 10% (w/v) honey water to the respective working concentration for the experiments. The concentration of a typical overnight culture was determined for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. by plating part of the overnight culture on agar plates and counting CFUs after 24 hr or 48 hr of growth at 30°C, for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. respectively. This yielded a concentration of 1.865 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">9</span></sup> ± 5.63 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">7</span></sup> (mean ± sd) bacteria per mL for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and 5.13 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">8</span></sup> ± 8.48 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">6</span></sup> (mean ± sd) bacteria for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="survival-experiments"> Survival experiments</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In a first survival experiment we tested whether the ability to perform acidopore grooming within the first 24 hr after ingestion of pathogen contaminated food provides a survival benefit for individual <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants. Ants from eight colonies were starved for 24–48 hr before start of the experiment, as described before, and then workers put singly in small petri dishes were either given access to 5 µL of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> contaminated 10% honey water (9.33 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">9</span></sup> bacteria/mL; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia+</em> ants: n = 127) or uncontaminated 10% honey water (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia-</em> ants: n = 135) for 2 min. Afterward, all ants were cold anaesthetized and approximately half of the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia+</em> and the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia-</em> ants (n = 65 and n = 69, respectively) immobilized in a pipetting tip, thus preventing acidopore grooming (FA- ants: n = 134) while the other half remained fully mobile (FA+ ants: n = 128). After 24 hr, FA- ants were freed from the pipetting tip to minimize stress. Mortality of the ants was monitored over 5 d (120 hr) every 12 hr providing no additional food, except the one time feeding of 5 µL contaminated or uncontaminated honey water at the start of the experiment. We chose to provide no additional food after the one time feeding at the beginning of the experiment, as an altered feeding behavior, that is, illness induced anorexia with known positive or negative effects on survival (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib65">bib65</a></cite>), might otherwise have influenced our results.</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In an additional survival experiment, we investigated whether the ability to acidify the crop lumen has the potential to limit oral disease transmission during trophallactic food transfer. To this end, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants from seven colonies were again starved, divided randomly in two groups (donor and receiver ants, each n = 322) and their gaster marked with one of two colors (Edding 751). Additionally, to prevent uptake of the poison, the acidopore opening of all receiver ants (receiver FA-) and half of the donor ants (donor FA-) was sealed with superglue, while the other half of the donor ants were sham treated (donor FA+) with a droplet of superglue on their gaster (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib140">bib140</a></cite>). We then paired these ants into two different donor-receiver ant pairs. Pairs with both donor and receiver ants having their acidopore sealed (donor FA- | receiver FA-) and pairs with only receiver ants having their acidopore sealed (donor FA+ | receiver FA-). Six hours after pairing, donor ants from both pairs were isolated and given access to 5 µl of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> contaminated 10% honey water (1.865 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">9</span></sup> bacteria/mL) for 12 hr. Thereafter donor ants were again paired with the respective receiver ants for 12 hr and all pairs filmed for the first 30 min (Logitech webcam c910). These videos were then analyzed for the duration of trophallaxis events donor-receiver ant pairs engaged in during the first bout of trophallactic food exchange. After this first feeding round, donor ants were fed in the same fashion, that is, isolation for 12 hr with access to <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> contaminated 10% honey water, every 48 hr, while they were maintained with the respective receiver ants for the rest of the time. This experimental design ensured that receiver ants were fed only through the respective donor ants with pathogen contaminated food. Survival of both, donor and receiver ants, was monitored daily for a total of 12 d.</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="bacterial-viability-and-growth-assays">Bacterial viability and growth assays</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">We tested the ability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. to withstand acidic environments in vitro, as well as their ability and the ability of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> to pass from the crop to the midgut in vivo when ingested together with food. In ants, gut morphological structures, that is, the infrabuccal pocket, an invagination of the hypopharynx in the oral cavity (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib40">bib40</a></cite>), and the proventriculus, a valve that mechanically restricts passage of fluids from the crop to the midgut (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib41">bib41</a></cite>), consecutively filter solid particles down to 2 µm (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib85">bib85</a></cite>) which would allow <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> (Ø: 0.5–0.8 µm, length: 0.9–2 µm, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib54">bib54</a></cite>), <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. (Ø: 0.4–1 µm, length: 0.8–2.5 µm, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib82">bib82</a></cite>), and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> (length: 1 µm, width: 0.35 µm, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib10">bib10</a></cite>) to pass. For the in vitro tests we incubated a diluted bacterial overnight culture (10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">5</span></sup> and 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">4</span></sup> CFU/ml for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp., respectively) in 10% honey water (pH = 5) and in 10% honey water acidified with commercial formic acid to a pH of 4, 3, or 2 for 2 hr at room temperature (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: n = 15 for all pH-levels, except pH = 4 with n = 13; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.: n = 10). Then we plated 100 µl of the bacterial solutions on agar-medium (LB-medium and GLY-medium for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp., respectively) and incubated them at 30°C for 24 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>) or 48 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.) before counting the number of formed CFUs. For the in vivo tests <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> ants from five (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.), four (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>) or from six colonies (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>) were starved as before and then individually given access to 5 µL of bacteria contaminated 10% honey water (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>: 1 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">7</span></sup> CFU/mL, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: 1 * 10<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">6</span></sup> CFU/mL) for 2 min. To assess the number of CFUs in the digestive tract, that is the crop and the midgut, ants were dissected either directly after feeding (0 hr; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: n = 60 workers; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>: n = 15 each), or at 0.5 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: n = 60; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>: n = 15 each), 4 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: n = 60; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>: n = 15 each), 24 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: n = 53; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>: n = 15 each) or 48 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>: n = 19; <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>: n = 15 each) after feeding. For dissection, ants were cold anesthetized, the gaster opened and the whole gut detached. The crop and the midgut were then separated from the digestive tract, placed in a reaction tube, mechanically crushed with a sterile pestle and dissolved in 100 µL (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>) or 150 µL (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>) phosphate buffered saline (PBS-buffer: 8.74 g NaCl, 1.78 g Na<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span data-itemtype="http://schema.org/Number">2</span></sub>HPO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span data-itemtype="http://schema.org/Number">4</span></sub>,2H<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span data-itemtype="http://schema.org/Number">2</span></sub>O in 1L MilliQ-water adjusted to a pH of 6.5). The resulting solutions were then thoroughly mixed, 100 µl streaked on agar-medium (LB-medium and GLY-medium for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em>/<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E.coli</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp., respectively) and incubated at 30°C for 24 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em>) or 48 hr (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp.), before counting the number of formed CFUs. No other bacteria (e.g. resident microbes) were apparent in terms of a different CFU morphology on the agar plates which agrees with the very low number of cultivable resident bacteria present in the midgut of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> (Stoll and Gross, unpublished results). This methodology cannot completely exclude that resident <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> or species of Acetobacteraceae might have biased our count data by adding a background level of CFUs at all timepoints or by adding random outlier CFUs at specific timepoints. Both, background level CFU numbers and random outlier CFUs should however not influence observed patterns over time. The timepoints of 0 hr, 0.5 hr, 4 hr, 24 hr, and 48 hr in in vivo bacterial growth assays were chosen according to literature describing passage of food from the crop to the midgut within 3–6 hr after food consumption in ants (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib25">bib25</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib79">bib79</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib78">bib78</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib67">bib67</a></cite>; <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib91">bib91</a></cite>). They should thus be representative of two time points before food passage from the crop to the midgut (0.5 hr and 4 hr) and two time points after food passage from the crop to the midgut (24 hr and 48 hr) together with the reference timepoint (0 hr).</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="food-passage-experiment"> Food passage experiment</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To estimate food passage from the crop to the midgut and hindgut of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> after feeding we performed the following experiment. We again took a cohort of ~100 workers out of one natal colony of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em>, starved them for 24 hr and then offered them 200 µL of a 1:1 honey-water mix with 50 mg of polymethylmethacrylate (PMMA, aka acrylic glass) particles (size ≤40 µm). Afterward, we dissected the digestive tract of three major and three minor workers at each of the timepoints 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 14 hr, 16 hr, 18 hr, 24 hr, and 48 hr after feeding and placed each under a microscope (Leica DM 2000 LED) to detect and count the number of particles via fluorescence in the crop, the midgut, and the hindgut.</p> <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="statistical-analyses"> Statistical analyses</h3> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">All statistical analyses were performed with the R statistical programming language (version 3.6.1, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib118">bib118</a></cite>). All (zero-inflated) General(ized) linear and mixed models and Cox mixed-effects models were compared to null (intercept only) or reduced models (for those with multiple predictors) using Likelihood Ratio (LR) tests to assess the significance of predictors. Pairwise comparisons between factor levels of a significant predictor were performed using pairwise post-hoc tests adjusting the family-wise error rate according to the method of Westfall (package ‘multcomp’, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib14">bib14</a></cite>). We checked necessary model assumptions of (zero-inflated) General(ised) linear and mixed models using model diagnostic tests and plots implemented in the package ‘DHARMa’ (<cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib58">bib58</a></cite>). Acidity of the crop lumen (log transformed pH to normalize data) and midgut lumen in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> was analyzed using linear mixed models (LMM, package”lme4’, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib6">bib6</a></cite>) including time since feeding (four levels: 0+4 hr, 0+24 hr, 0+48 hr, 48h+4 hr; <a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1a</a>), ant manipulation (two levels: FA+ and FA-, that is ants with and without acidopore access; <a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1b</a>) or digestive tract part (four levels: crop, midgut position 1, midgut position 2, midgut position 3, midgut position 4; <a href="#fig1s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 1</a>) as predictors and natal colony as a random effect. Due to non-normality and heteroscedasticity, the acidity of the crop lumen in the seven formicine ant species other than <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> (<a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1c</a>) was analysed using per species Wilcoxon rank-sum tests with ant manipulation (FA+ and FA-) as predictor. The frequency of acidopore grooming in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> upon feeding different types of fluids was analyzed using Generalized linear mixed models (GLMM, package”lme4’, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib6">bib6</a></cite>) with negative binomial errors and type of fluid (three levels: unfed, water-fed, or 10% honey water fed) as predictor and natal colony as random effect (<a href="#fig1s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 2</a>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Survival data were analysed with Cox mixed effects models (COXME, package ‘coxme’, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib138">bib138</a></cite>). For the survival of individual ants (<a href="#fig3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3</a>), ant treatment (four levels: <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia-</em> | FA-, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia-</em> | FA+, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia</em>+ | FA-, <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Serratia+</em> | FA+) was added as a predictor and the three ‘blocks’ in which the experiment was run and the colony ants originated from, were included as two random intercept effects. For the survival of donor-receiver ant pairs (<a href="#fig4" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 4</a>), ant treatment (four levels: donor FA+, donor FA-, receiver FA+, receiver FA-) was included as a predictor and the three ‘blocks’ in which the experiment was run, the colony ants originated from, and petri dish in which donor and receiver ants were paired, were included as three random intercept effects. Survival of receiver ants was right censored if the corresponding donor ant died at the next feeding bout (right censoring of both donor and receiver ants in one pair upon death of one of the ants yielded statistically the same result: COXME, overall LR χ<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span data-itemtype="http://schema.org/Number">2</span></sup> = 60.202, df = 3, p<0.001; post-hoc comparisons: receiver FA- versus donor FA-: p=0.388, all other comparisons: p<0.001). The duration of trophallaxis events (square-root transformed to normalize data) between donor-receiver ant pairs was analysed using a linear mixed model with ant pair type (two levels: donor FA+ | receiver FA- and donor FA- | receiver FA-) as predictor and the three ‘blocks’, in which the experiment was run and the colony ants originated from as random effect (<a href="#fig4s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 4—figure supplement 1</a>).</p> <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Bacterial growth in vitro was analysed separately for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. using Generalized linear models (GLM) with negative binomial errors and pH as predictor, excluding pH levels with zero bacterial growth due to complete data separation (<a href="#fig2s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 2</a> and <a href="#fig5s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5—figure supplement 1</a>). Relative values shown in <a href="#fig2s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 2</a> and <a href="#fig5s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5—figure supplement 1</a> were calculated by dividing each single number of formed CFUs at the different pH-values through the mean of formed CFUs at pH 5. Bacterial viability in vivo within the digestive tract of <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">C. floridanus</em> over time was analysed separately for the crop and midgut for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">S. marcescens</em> and <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Asaia</em> sp. (<a href="#fig2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2</a> and <a href="#fig5" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5</a>, respectively) and for <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> (<a href="#fig2s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 2</a>). Zero-inflated generalized linear mixed models with negative binomial errors (package ‘glmmTMB’, <cite itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a href="#bib17">bib17</a></cite>) were used to model CFU number, with time after feeding as fixed predictor and ant colony as random effect, except for the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">E. coli</em> model in the crop where colony was included as fixed factor as the model did not converge with colony as a random factor. Timepoints with zero bacterial growth were again excluded in the models. Relative CFU values shown in <a href="#fig2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2</a>, <a href="#fig5" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5</a>, and <a href="#fig2s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2—figure supplement 2</a> were calculated by dividing single CFU-values through the mean of CFU-values at timepoint 0 hr in the crop. Proportions and percentages of relative CFU change in the text are based on these relative CFU values.</p> </article> </main> </body> </html>