Defensive symbiont genotype distributions are linked to parasitoid attack networks

Defensive symbiont genotype distributions are linked to parasitoid attack networks | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Defensive symbiont genotype distributions are linked to parasitoid attack networks View ORCID Profile Taoping Wu , Anoushka A. Rodrigues , Tom Fayle , View ORCID Profile Lee M. Henry doi: https://doi.org/10.1101/2024.07.24.604610 Taoping Wu 1 School of Biological and Chemical Sciences, Queen Mary University of London , London, E1 4NS, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Taoping Wu Anoushka A. Rodrigues 1 School of Biological and Chemical Sciences, Queen Mary University of London , London, E1 4NS, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tom Fayle 1 School of Biological and Chemical Sciences, Queen Mary University of London , London, E1 4NS, United Kingdom 2 Biology Centre of the Czech Academy of Sciences, Institute of Entomology , Ceske Budejovice, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lee M. Henry 1 School of Biological and Chemical Sciences, Queen Mary University of London , London, E1 4NS, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lee M. Henry For correspondence: l.henry{at}qmul.ac.uk Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Facultative symbionts are widespread in arthropods and can provide important services such as protection from natural enemies. Yet what shapes associations with defensive symbionts in nature remains unclear. Two hypotheses suggest that either interactions with antagonists, or host plants, may explain the prevalence of symbionts through shared selective pressures and routes of horizontal transmission. Here we investigate the factors driving similarities in the Hamiltonella defensa symbiosis shared among host species within field collected aphid communities. We show that, Hamiltonella’s genotype distribution strongly aligns with sharing the same parasitoids, rather than host plants, highlighting parasitoids as a key selective agent shaping the symbiosis across host species. Our data indicates parasitoid host-specificity drives the prevalence of specific aphid- Hamiltonella associations, suggesting defensive symbioses are maintained by the selective pressure imposed by dominant parasitoid species. These findings underscore the importance of interactions with natural enemies in explaining patterns of defensive symbiosis in nature. INTRODUCTION Heritable bacterial symbionts are widespread in insects and often confer ecologically important traits to their hosts. Many insects harbour vertically transmitted obligate symbionts that have enabled expansions into novel feeding niches by provisioning their hosts with essential nutrients ( Cornwallis et al . 2023 ; Jackson et al . 2022 ). Even more widespread are heritable facultative symbionts, which are not essential for hosts but can provide important context-specific benefits, such as protection from biotic or abiotic stresses ( Heyworth et al . 2020 ; McLean & Godfray 2015 ; Tougeron & Iltis 2022 ; Wu et al . 2022 ). Facultative symbionts can also be horizontally transferred between hosts ( Henry et al . 2015 ; Russell et al . 2003 ), and tend to be non-randomly distributed among plant-adapted insect populations and species (e.g. Henry et al . 2015 ; Henry et al . 2022 ; Jackson et al . 2023 ; Toju & Fukatsu 2011 ; Wu et al . 2022 ), although the reasons for this are currently unknown. This has led researchers to propose that facultative symbionts may provide benefits to populations in certain ecological niches, with high frequencies of symbiont carriage due to the acquisition and spread of symbionts that confer local adaptations ( Jaenike 2012 ). However, the role of facultative symbionts in insect ecology has been hampered by our limited understanding of symbiont exchange networks and the agents of selection shaping their populations. Insect facultative symbionts are often associated with providing protection against natural enemies (e.g. Oliver et al . 2003 ; Vorburger et al . 2010 ; Xie et al . 2014 ; Oliver & Perlman 2020 ; Zhao et al . 2023 ). While laboratory studies have provided valuable insight into defensive symbioses, our understanding of their network ecology in the wild is limited. Defensive symbionts are often maintained at intermediate frequencies in host populations (e.g. Ferrari et al . 2012 ; Osaka et al . 2010 ; Henry et al . 2013 , 2015 ), suggesting a range of factors influence their spread and maintenance ( Oliver et al . 2008 ; Smith et al . 2021 ). Selective forces such as the cost-benefit balance of maintaining symbionts in different ecological scenarios, as well as non-selective factors such as transmission rates, migration, and drift likely shape defensive symbiont distributions in host populations (reviewed in Vorburger 2022 ). Moreover, the horizontal transfer of facultative symbionts among host species complicates our understanding of how defensive symbioses function within insect communities, particularly those facing diverse natural enemies. The function of defensive symbioses is likely to have important implications for the dynamics of both natural ecosystems and agricultural landscapes, in which aphids are an important pest group (e.g. Leclair et al . 2021 ) Some of the best-known examples of defensive symbioses have emerged from the bacterial symbionts of aphids. In addition to their obligate nutritional symbiont, Buchnera aphidicola , aphids harbour an array of facultative symbiont species, several of which can confer protection against natural enemies ( Ferrari et al . 2004 ; Oliver et al . 2003 ). Among these, Hamiltonella defensa , hereafter Hamiltonella , stands out for its widespread presence in aphid populations and its ability to protect aphids from parasitoid wasp attack. In nature, aphid species and plant-adapted aphid ‘biotypes’, tend to be associated with only a few closely related Hamiltonella strains ( Henry et al . 2013 ; Wu et al . 2022 ), however the reason for this is currently unclear. Hamiltonella is strongly associated with aphids that feed on certain host plants, suggesting interactions with plants may shape its distribution ( Gimmi et al . 2024 ; Henry et al . 2013 , 2015 ). However, different strains of Hamiltonella can also protect against different species of parasitoid wasps ( McLean & Godfray 2015 ). In nature, some aphids have retained Hamiltonella strains that provide strong protection against their main parasitoid species ( Gimmi & Vorburger 2024 ; Wu et al . 2022 ), suggesting antagonists may contribute to the symbiont’s distribution in nature. In addition, phylogenetic studies have shown that Hamiltonella can horizontally transfers between unrelated aphid species ( Henry et al . 2013 ; Wu et al . 2022 ), although how transfer occurs in nature is unclear. Laboratory studies have shown that Hamiltonella can be transferred between hosts by parasitoids through a contaminated ovipositor ( Gehrer & Vorburger 2012 ), but also via plant phloem ( Li et al . 2018 ), suggesting that both mechanisms may contribute to the dynamics of symbiont acquisition and transmission in nature. However, the relative importance of plant and parasitoid interactions in shaping Hamiltonella ’s presence in aphid communities remains unclear. In this study, we analyse data from 1257 aphid samples to investigate ecological correlates linked to Hamiltonella ’s distribution. Specifically, we ask whether Hamiltonella strains found in different aphid species are best explained by sharing parasitoids or host plants to shed light on the drivers of defensive symbiont dynamics in nature. MATERIALS AND METHODS Aphid-parasitoid sampling and identification In 2021 and 2022, live and ‘mummified’ aphids were collected from 17 plant species in 26 locations across Greater London (Tables S1, S2), focusing on aphid species known to harbour Hamiltonella (from Henry et al . 2015 ; Wu et al . 2022 ). Aphids were collected by beating plants over a tray or manually from leaves and stems. To avoid resampling the same aphid clone or mummies from the same parasitoid, collections from the same plant species were spaced at least 10 meters apart. Additionally, 241 plant-aphid- Hamiltonella samples from a previous study ( Wu et al . 2022 ) were included to control for potential sampling biases due to only sampling parasitised aphids. For robustness, we analyzed both the full data set of 1257 individuals and a conservative data set of 1016 samples collected in 2021 and 2022 from mummified aphids. Aphid-parasitoid associations were identified using two methods: 1) Illumina amplicon sequencing of collected mummies, and 2) culturing live aphids until mummies formed and sequencing the emerged wasps. Mummified aphids were preserved in 70% ethanol for Illumina amplicon sequencing of the Cytochrome Oxidase subunit I (COI) gene to identify both parasitoid and aphid species. Live aphids, morphologically identified to species, were kept at 15°C with a 16h:8h light cycle for two weeks on host plant cuttings to harvest additional parasitoids. Emerging parasitoids were isolated, COI barcoded, and Sanger sequenced at Source Bioscience Ltd (London, UK) for identification. Morphologically identified aphids were also confirmed by Sanger sequencing. DNA extractions were performed using DNeasy Blood and Tissue Kits (QIAGEN, Venlo, Netherlands). DNA from mummies was normalized to 5 ng/μL. A 407 bp COI gene region was amplified and sequenced using CS1/CS2 tagged primers and a modified PCR protocol (Table S5). Tagged PCR products were sequenced at the London Genome Centre. We sequenced 1108 mummies in two Illumina MiSeq runs using paired-end 300 bp reads: 370 samples in 2021 with 30,743 reads per sample, and 738 samples in 2022 with 26,281 reads per sample. Sequencing data was analyzed using the DADA2 pipeline ( Callahan et al . 2016 ), with reads trimmed for quality, deduplicated, and chimeric sequences removed. Species were identified by comparing COI sequences to GenBank using BLASTn. Sequences were aligned using MUSCLE aligner in MEGA11 ( Tamura et al. 2021 ). Out of 1108 sequenced mummies, 833 yielded COI data for both parasitoid and aphid, with 129 detecting Hamiltonella for MLST. An additional 183 parasitoid samples identified by Sanger sequencing brought the total to 1016 parasitoid-aphid- Hamiltonella samples. Transient associations, where single aphids were collected from unrecognised host plants, were removed from the data set. Non-mummified samples, and those where Hamiltonella was not detected, were scored as having ‘No Association’ for parasitoid or symbiont and removed from corresponding analyses. Combined, these data sets resulted in 1257 samples for tripartite analyses. Hamiltonella strain identification Samples from 2021/2022 were screened for Hamiltonella using diagnostic PCR targeting the 16S rRNA gene ( Niepoth et al . 2018 ) (Table S5). Positive samples were genotyped using Multilocus Sequence-Typing (MLST) of four bacterial housekeeping genes: accD, hrpA, murE, recJ ( Degnan & Moran 2008 ) (Table S5). PCR products were Sanger-sequenced in one direction and aligned using MUSCLE aligner in MEGA11 ( Tamura et al . 2021 ). Hamiltonella sequences from aphids collected between 2011 and 2019 ( Wu et al . 2022 ) were included in the alignment. The four genes were concatenated for analyses. Clustering of operational taxonomic units COI gene sequences exhibiting over 99% similarity were clustered into a single Operational Taxonomic Unit (OTU). Species identifications for OTUs were based on the closest matching sequences in GenBank, with those >99% similarity assigned morphospecies name (sp1, sp2 etc). Multiple parasitoid OTUs matching the same species were designated as separate lineages (e.g., ‘clade 1’, ‘clade 2’) in analyses. For Hamiltonella , each MLST type was considered a separate OTU and referred to as a ‘strain’. Phylogeny reconstruction Phylogenetic trees for aphids (COI), parasitoids (COI), and Hamiltonella (MLST) were constructed using the Maximum Likelihood method on the ATGC bioinformatics platform, visualized and rooted using iTOL software. Adelges cooleyi , Formica fusca , and Hamiltonella strain MEAM1 of Bemisia tabaci were used as roots for aphid, parasitoid, and symbiont phylogenies, respectively. Bayesian Information Criterion (BIC) was used for model selection through Smart Model Selection (SMS). Trees were generated in PhyML 3.0 ( Guindon et al . 2010 ) using 100 bootstrap replicates. Network visualizations and specialization tests Network visualizations were conducted using R v4.2.2 in RStudio. The aphid- Hamiltonella -plant and aphid- Hamiltonella -parasitoid interactions ( Fig. 2 ) were mapped using the ‘igraph’ package ( Csárdi et al . 2024 ) with the Fruchterman-Reingold layout. Networks showed similar patterns when reduced to data collected in 2021 and 2022. Bipartite plots ( Fig. 4 ) were constructed using the ‘plotweb’ command in the ‘bipartite’ package ( Dormann et al . 2008 ), assigning Hamiltonella strains to parasitoids proportionally. Hamiltonella aphid interactions matrix was plotted using the ‘ape’ package ( Paradis & Schliep 2019 ) as in Wu et al . (2022) . Network specialization was tested using the H2’ index ( Blüthgen et al . 2007 ) by compare observed H2’ values with those from the null models using 1000 randomizations. Statistical analysis Statistical analyses were conducted in R v4.2.2. Relationships between Hamiltonella strain diversity and parasitoid or plant diversity were tested using a linear model in the ‘lme4’ package ( Bates et al . 2015 ), visualised in ggplot2 (Hadley Wickham 2016 ) and ‘igraph’ ( Csárdi et al . 2024 ). Data were rarefied (n=1000 randomisations) to account for differences in sample sizes using the ‘rrarefy’ function in ‘vegan’. Diversity indexes were calculated using custom scripts. Mantel tests compared dissimilarity matrices of parasitoid, plant, and Hamiltonella communities using the Bray-Curtis dissimilarity matrix in the ‘vegan’ package with 9999 permutations. Aphid species lacking data for either parasitoids or Hamiltonella strains were excluded from analyses to prevent missing data from impacting results. This ensured accurate interpretation of relationships among aphids, parasitoids, and Hamiltonella strains. RESULTS Charactering the parasitoid and plant associations of aphids Our analysis of the 1257 samples identified 45 parasitoid species and 36 Hamiltonella strains associated with 31 aphid species collected from 62 species of plants ( Fig. 1 , Table S1 and Table S2). A phylogeny based on the parasitoid COI gene revealed that the parasitoid species belong to 40 species (5 sub-species) from 12 genera and 2 Hymenopteran families, the Braconidae and Chalcididae. However, the vast majority (43/45) of aphid parasitoids were Braconids (Fig. S1). Most parasitoid species (65%) were recovered from more than one aphid species. However, most parasitoids were primarily associated with a single aphid species or genus (although the host species varied between parasitoids) with only intermittent use of other aphid species ( Fig. 1 ). The majority (14/22 species, 64%) of aphid species were attacked by a single dominant parasitoid that accounted for >75% of parasitism for each aphid species, and most aphid species (18/31 species, 58%) fed on a single dominant species of plant (although again, the dominant parasitoid and plant species varied between aphid species). There were also numerous cases where multiple aphid species share the same host plant or parasitoid species ( Fig. 1 , Table S1). This demonstrates that this is an appropriate system to test whether the sharing of plants or parasitoids is linked to the sharing of Hamiltonella strains across aphid species. Download figure Open in new tab Figure 1: Relative frequency of parasitoid and host plant associations across aphids. Each bar represents an aphid species with different colours denoting the relative abundance of (A) different parasitoid species attacking the aphids and (B) different plant genera on which the aphids feed. Bars are organised according to the similarity of parasitoid/host plant communities as determined by Hierarchical Clustering. For visualisation purposes, different OTUs, or ‘clades’ belonging to a single parasitoid species were merged into a single colour. Hamiltonella diversity across aphid species We were able to amplify Hamiltonella from 129 parasitoid mummies (Table S2). Genotyping the symbionts revealed 7 new Hamiltonella strains associated with aphids. By placing the new stains in a Hamiltonella -aphid phylogenetic matrix with previously published stains ( Wu et al ., 2022 ) we revealed that the newly identified strains tend to be associated with a single aphid species (Fig. S2). For example, Hamiltonella strain N_1_88 was only found in Periphyllus lyropictus and was found at relatively high frequencies (47%, 16 out of 34) in this aphid. However, we also found new cases of the same symbiont strains being shared by distantly related host species. For example, the aphids Cavariella theobaldi and Macrosiphum funestrum were both found with Hamiltonella strain N_2_65 and the aphids Aphis fabae and M. funestrum were both found harbouring strain N_4_75. In total, 15 out of 36 (42.6%) Hamiltonella strains were found across multiple aphid species. Hamiltonella strain 231 and strain 1485 are particularly noteworthy in their distributions; strain 231 was found in 14 aphid species across 8 genera and strain 1485 in six aphid species belonging to 3 genera. Our analysis also revealed several cases where a single aphid species was associated with two or more unrelated Hamiltonella symbiont strains. For example, A. fabae , P. lyropictus , M. funestrum , Metopolophium dirhodum , Hyperomyzus lactucae , and the Medicago biotype of A. pisum were all associated with multiple unrelated Hamiltonella strains that they carried at relatively high frequencies (Fig. S2). We tested whether aphids associated with greater Hamiltonella diversity were associated with greater parasitoid or plant diversity using both rarefied and raw data sets (Table S3). Irrespective of the data set used, there was no correlation between Hamiltonella strain diversity, and the diversity of parasitoid or plant species associated with aphids (Fig. S3, Table 1 ). View this table: View inline View popup Download powerpoint Table 1: General linear model (GLM) of the association between Hamiltonella strain diversity and parasitoid/host plant diversity in aphids. Three data set were tested, including the full data set, rarefied (n = 7) data set and our conservative data set based on mummies only. Aphid- Hamiltonella associations are correlated with parasitoid attack networks Comparing Bray-Curtis indexes, we found that aphid species that shared the same parasitoid species tended to harbour the same Hamiltonella strains ( Fig. 2A ). However, aphid pairs that fed on the same species of plants had fewer incidences of sharing Hamiltonella strains ( Fig. 2B ). Using Mantel tests, we confirmed that there was a strong positive correlation between the similarity of Hamiltonella diversity shared by aphids and their parasitoid communities (Mantel test: r=0.294, p=0.0083**, Fig. 3A ), but not their food plants (Mantel test: r=0.036, p=0.209, Fig. 3B ). For robustness, we also analysed the data using only the samples collected in 2021 and 2022 and found the results were largely similar (Fig. S4 and S5). Download figure Open in new tab Figure 2: Parasitoid- Hamiltonella and plant- Hamiltonella network similarity of aphids Grey lines connect aphid species that share similar Hamiltonella and (A) parasitoid species or (B) plant species. The thickness of each line corresponds to the sum of the Bray-Curtis similarity values of parasitoids/plants and Hamiltonella communities (Table S4). The size of each node reflects the number of Hamiltonella samples collected for each aphid species, while the colour denotes different aphid-plant classification: pink for tree-dwelling aphids, yellow for predominantly grass-dwelling aphids (although some host alternate), and green for herb-dwelling aphids. Red circles highlight predominant parasitoid and host plant associations (at > 0.1 Bray-Curtis similarity) that also share Hamiltonella strain(s). Parasitoid species and host plant genera (or families) are indicated in black squares. Download figure Open in new tab Figure 3: Hamiltonella -parasitoid and Hamiltonella -plant community similarity across pairs of aphid species. Each point represents the community similarities of a pair of aphid species. The x-axis represents the similarity in their (A) parasitoid, or (B) host plant community composition, while the y-axis represents the similarity in their Hamiltonella strain composition. Pearson correlation coefficients (r) and p-values ( p ) from Mantel tests are given in the upper right of each panel. Regression lines with 95% confidence intervals are plotted for visualisation purposed only, but not used in statistical analyses. Within the parasitoid-aphid- Hamiltonella data, we identified 85 pairs of aphid species that shared Hamiltonella strains; among these, 47 pairs (55.3% of pair-wise comparisons) exhibited varying degrees of similarity in their parasitoid compositions (Table S4). 15 out of 19 (78.9%) aphid species sharing Hamiltonella strains could be linked by sharing at least one parasitoid species (Table S4). This is particularly evident in the Macrosiphum genus, which has the highest similarity in parasitoid species that attack them and tend to harbour the same Hamiltonella strains (average parasitoid Bray-Curtis similarity: 0.244, average Hamiltonella Bray-Curtis similarity: 0.423, Table S4). Several aphids that feed on grasses, including Utamphorophora humboldti , M. dirhodum , Sitobion avenae and Sitobion fragariae showed relatively high degree of parasitoids (Bray-Curtis = 0.156) and Hamiltonella similarity (Bray-Curtis = 0.074) with each other, as well as with Macrosiphum aphids (average parasitoid and Hamiltonella Bray-Curtis = 0.041 and 0.131, respectively, Table S4). The two Caveriella species, C. theobaldi and C. pastinacae , are also predominately attacked by the same parasitoid (Parasitoid Bray-Curtis = 0.33) and have evidence of sharing the same Hamiltonella strain 231 (Parasitoid Bray-Curtis = 0.11), but also feed on the same host plant, Heracleum sphondylium . The plant-aphid- Hamiltonella data revealed 138 pairs of aphid species that shared symbiont strains; yet only 20 pairs (14.5% of pair-wise comparisons) had similarities in host plant species ( Fig. 3B , Table S4). Furthermore, 8 aphid species, A. pisum, Aphis ruborum, Aphis sambuci, Macrosiphum albifrons, P. lyropictus, Uroleucon escalantii, Uroleucon sonchi, Wahlgreniella nervata , shared Hamiltonella strains with other aphid species but were not found sharing any host plants in our data set. Unlike the parasitoid-aphid- Hamiltonella interactions that linked all correlated aphid species together, the plant-aphid- Hamiltonella interaction formed three groups: i) 3 of 4 aphids feeding on Rubus are grouped with aphids that feed on grasses (Poaceae) through the host-plant alternating Sitobion fragariae , which are then weakly connected to 3 Macrosiphum species feeding on Geum ; ii) 2 of 3 species on Sonchus share some similarity; and iii) the two Cavariella species that feed on Heracleum share plants, parasitoids, and Hamiltonella strains ( Fig. 2 ). Notably, there were 4 aphid species, P. lyropictus , U. sonchi , W. nervata and A. ruborum , that shared Hamiltonella strains but did not share the same parasitoids or host plants. This indicates that there were still connections in the symbionts’ distribution that cannot be explained by the plants or parasitoids identified in our data set. Specialised parasitoid-aphid- Hamiltonella associations Our analyses revealed high degrees of specialisation in all interaction networks, indicating aphids, parasitoids, and symbiont strains tend to interact with a limited number of partners ( Fig. 4 ). Specialization (as measured using H2’) was highest for parasitoid-aphid networks (Standardised Effect size (SES) = 153.3), but also high for both Hamiltonella -aphid (SES = 48.3) and the generated Hamiltonella -parasitoid (SES = 34.0) networks. Most aphid species harboured a single dominant Hamiltonella strain and were primarily targeted by a specific parasitoid species ( Fig. 4A & 4B). For example, in the Macrosiphum genus, particularly Macrosiphum gei , M. euphorbiae , and Macrosiphum hellebori , all species predominantly harboured Hamiltonella strain 231 and were mainly attacked by a single parasitoid species, Aphidius rhopalosiphi clade 1. Notably, A. rhopalosiphi clade 1 was the most generalist parasitoid in our study, attacking 8 aphid species belonging to 4 genera; 6 of the species carried Hamiltonella strain 231. In addition to A. rhopalosiphi clade 1, Macrosiphum aphids were attacked by a similar group of parasitoids at lower incidences, including A. rhopalosiphi clades 2 and 3, and several species from the Aphidius , Praon , and Ephedrus genera ( Fig. 4B ). Aphis fabae also maintained diverse connections in that it shared Hamiltonella strains and parasitoids attacking it at relatively low frequencies with 10 other aphid species from 4 genera, including Acyrthosiphon , Cavariella , Macrosiphum , Metopolophium , and Hyperomyzus ( Fig. 4 , Table S1 & S5). Conversely, there were also aphid species, such as Drepanosiphum platanoidis and Macrosiphoniella artemisiae , that harboured a unique cluster of Hamiltonella strains and were each attacked either primarily or exclusively by a single parasitoid species. The parasitoid- Hamiltonella network also revealed several cases where parasitoids had particularly strong associations with certain symbiont strains, such as A. rhopalosiphi and A. ervi , which are linked to Hamiltonella strains 231 and 2578, respectively. Download figure Open in new tab Figure 4: Bipartite parasitoid, aphid, and Hamiltonella interaction networks. Networks depict (A) Hamiltonella strain (top) – aphid species (bottom), (B) parasitoid species (top) – aphid species (bottom), and (C) parasitoid species (top) – Hamiltonella strain (bottom) interactions. The parasitoid- Hamiltonella network were generated by assigning Hamiltonella “individuals” in each aphid species to parasitoids attacking them, proportional to the frequency of attacks across different parasitoid species. Grey lines connect interacting species and strains, and the width of the line indicates the frequency of interaction between pairs. The width of the black bars denotes the relative abundance of individual aphids, parasitoids, and Hamiltonella stains used in each bipartite network, because these were assessed using different sets of samples. DISCUSSION We demonstrate that natural enemy attack networks are linked to the distribution of defensive symbiont strains within an insect community. Population surveys, including ours, show that facultative symbionts providing protection tend to be non-randomly distributed across aphid species and plant-adapted populations; only certain species and populations harbour them, often with few symbiont strains at high frequency ( Henry et al . 2013 , 2015 ; Wu et al . 2022 ). We show aphids are typically attacked by a single dominant parasitoid species, and aphid species sharing the same parasitoids, rather than food plants, tend to carry the same strains of Hamiltonella . This suggests that interactions with parasitoids play a key role in the spread and maintenance of this defensive symbiosis within aphid communities. Parasitoids as selective agents shaping Hamiltonella ’s distributions Parasitoids impose strong selective pressure for the evolution of resistance in aphids. We show each aphid species is frequently attacked by a single dominant parasitoid, demonstrating a high degree of host specialization. We suggest parasitoid host-specificity may shape Hamiltonella distributions across aphid species. Laboratory studies have shown Hamiltonella strains vary in their degree of protection and specificity against different parasitoid species ( Cayetano et al . 2015 ; Martinez et al . 2016 ). For example, McLean & Godfray (2015) found Hamiltonella strains associated with pea aphids on Lotus plants protect against Aphelinus abdominalis , but not Aphidius ervi , while strains on Medicago plants show the opposite trend. Frequent attack by a single parasitoid species likely explains why aphids carry a single or few closely related Hamiltonella strains, as these provide strong protection against their main natural enemy. Laboratory studies support this, showing attack by a single parasitoid species can lead to aphids carrying a single highly protective symbiont strain ( Hafer-Hahmann & Vorburger 2020 ). Moreover, studies have found aphid species often carry Hamiltonella strains in nature that confer strong protection against their most common parasitoid ( Gimmi & Vorburger 2021 ; Wu et al . 2022 ). Strikingly, we reveal that phylogenetically unrelated aphids attacked by the same parasitoids also carry the same Hamiltonella strains (based on 4 MLST genes). This suggests aphids retain similar symbiont strains to protect against a shared enemy. While the molecular mechanism of protection isn’t fully resolved, the genomes of most Hamiltonella contain a toxin-encoding bacteriophage known as APSE that is likely involved ( Lynn-Bell et al . 2019 ). Variation in APSE’s toxin genes is thought to underlie the degree of protection against the parasitoid Aphidius ervi ( Oliver & Higashi 2019 ). It would be in interesting to know whether APSE toxin variability also explains protection against different parasitoid species. Although parasitoid and Hamiltonella community compositions were similar, there was no correlation between parasitoid species diversity and Hamiltonella strain diversity in aphids. Increased parasitoid diversity may not lead to increased symbiont diversity if there is a cost to resistance ( Hafer-Hahmann & Vorburger 2024 ). Furthermore, factors such as transmission efficiency, host-symbiont compatibility, or interactions with other microbes, pathogens, or the environment, may also impact Hamiltonella diversity ( Carpenter et al . 2021 ; Dykstra et al . 2014 ; Niepoth et al . 2018 ; Weldon et al . 2020 ; Goldstein et al . 2023 ). We also did not consider genotype diversity within parasitoid species, which may influence symbiont diversity associated with an aphid species ( Hafer-Hahmann & Vorburger 2020 ). Influence of plants on Hamiltonella ’s distribution Studies show facultative symbionts occur more frequently in insect populations on certain plants, suggesting plant interactions may shape their distributions ( Ferrari et al . 2004 ; Henry et al . 2013 , 2015 ; Toju & Fukatsu 2011 ; Tsuchida et al . 2002 ). Examples include pea aphid biotypes on Medicago sativa , Lotus pedunculatus , and Ononis plants carrying biotype-specific Hamiltonella strains, and A. fabae populations feeding on different plants differing in Hamiltonella carriage ( Gimmi et al . 2024 ; Henry et al . 2013 , 2015 ). However, our results suggest there is no link between host plant sharing and the sharing of Hamiltonella strains in aphids. This indicates that plants have a limited role in shaping Hamiltonella distribution, at least among aphid species. Aphids on different plants might attract different parasitoid species due to changes in plant volatile profiles or Hamiltonella itself may modifying plant volatiles to attract different parasitoid species ( Ahmed et al . 2022 ; Ali et al . 2022 ; Frago et al . 2017 ), leading to plant associated Hamiltonella strains. It would be interesting to determine if plant-adapted aphid biotypes are attacked by different parasitoid species, as this may explain their tendency to carry different Hamiltonella strains. Ecological vectors of Hamiltonella transmission Hypotheses explaining horizontal transmission of facultative symbionts among insects include natural enemy and plant sap transmission. Natural enemies might pick up and transmit symbionts via contaminated mouthparts or ovipositors ( Ahmed et al . 2015 ; Gehrer & Vorburger 2012 ; Kaech & Vorburger 2021 ; Soleimannejad et al . 2023 ; Tzuri et al. 2021 ), while plant sap transmission involves symbionts being released into plant sap and acquired by another insect ingesting it ( Chrostek et al . 2017 ; Li et al . 2018 ; Pons et al . 2019 ). We show that the same Hamiltonella strains occur in distantly related aphids attacked by the same parasitoid species. Moreover, aphids carrying low frequencies of Hamiltonella strains typically found in other aphids, are often also attacked by their parasitoids at low incidences. This suggests parasitoids may be a vector of symbiont transmission in wild aphid populations. Laboratory studies have shown Hamiltonella can be transmitted by parasitoids in A. fabae , with transmission success depending on symbiont titre and haplotype ( Kaech & Vorburger 2021 ). The likelihood of symbiont transfer can also be influenced by the relatedness of the hosts, and the symbionts they previously harboured ( Łukasik et al . 2015 ; McLean et al . 2019 ). Parasitoids have also been shown to transfer facultative symbionts in other insects, such as Myzus persicae and Bemisia tabaci , and in house flies, Musca domestica ( Ahmed et al . 2015 ; Soleimannejad et al . 2023 ; Tzuri et al . 2021 ). In contrast, we find little evidence that sharing the same host plants leads to sharing Hamiltonella strains. Plant-mediated horizontal transfer has been reported in several sap-feeding insects under laboratory conditions ( Caspi-Fluger et al . 2012 ; Gonella et al . 2015 ; Li et al . 2017 ), including Hamiltonella , where plant transfer has been shown in Sitobion miscanthi feeding on wheat ( Li et al . 2018 ). We find that when aphid species do share the same food plant and Hamiltonella strains, they also share the same parasitoids. Moreover, there are no cases where plant sharing alone explains Hamiltonella ’s distribution, even in aphids that have a strong similarity in plant use, and do not share parasitoids. This suggests horizontal transfer by plants is either infrequent in aphids, or potentially only occurs in certain plants. It is also possible that selection from parasitoids may rapidly purge plant transferred symbiont strains from aphid populations, thereby contributing to the observed pattern. However, in most cases where Hamiltonella strains are shared between aphids it cannot be explained by plants, suggesting if it does occur it is of limited importance. Facultative symbionts as a reservoir of adaptations in insect defence The non-random distribution of facultative symbionts in insects has puzzled scientists, especially in bacteria known to provide hosts with protection. Our results suggest that selection from natural enemies is key in shaping the distribution of defensive symbionts in aphids and potentially other insects. The link between Hamiltonella strains and parasitoid networks supports the idea that coevolutionary dynamics has led to host specialisation in natural enemies that has been largely mediated by the symbiont ( Vorburger 2022 ). This may be due to symbiont-conferred defences being more specific than host-encoded defences, allowing hosts to tailor defences towards specific enemies through symbiont acquisitions. Specificity in symbiont-mediated protection has been reported in several defensive symbioses ( Higashi et al . 2023 ; Łukasik et al . 2013 ; Mateos et al . 2016 ), and is particularly evident in Hamiltonella ( McLean & Godfray 2015 ; Rouchet & Vorburger 2012 ; Wu et al . 2022 ). Bacteria have the potential to evolve more rapidly than the host’s genome, particularly when genes involved in protection are located on mobile genetic elements, such as the diverse toxin genes contained on Hamiltonella ’s APSE phage. Parasitoids can become resistant to the presence of defensive symbionts (e.g. Dion et al . 2011 ; Oliver et al . 2008 ). The arsenal of defences carried by Hamiltonella ’s APSE phages, in combination with their potential to mobilise, may be the crucial component in winning the evolutionary arms race against parasitoids, leading to the widespread distribution of the symbiont in aphids. However, the selection from Hamiltonella on specific natural enemies may also disrupt host specialization causing parasitoid to switch hosts to avoid highly protective symbiont strains, or provide an advantage to secondary parasitoids that attack a host at lower frequencies (e.g. McLean & Godfray 2017 ). It has been suggested that horizontal transfer of defensive symbionts is rare enough that different host species carry distinct symbiont communities ( Vorburger 2022 ). Our results suggest otherwise, as aphids attacked by the same parasitoid species tend to harbour related Hamiltonella strains. This suggests a more dynamic relationship where symbionts are maintained in host populations, at least across generations, at time scales that are relevant to selection from parasitoids. However, a finer-scale genetic analysis of shared symbionts and phages is needed to confirm this. Nonetheless, horizontal transfer of symbionts by parasitoids, even if rare, likely contribute to the genetic similarly of Hamiltonella strains occurring in aphids that share the same parasitoids and provides an important source of incoming symbiont variants for selection to act upon. Future studies are needed to assess whether symbiont transfer is rapid enough to counteract changes in parasitoid communities, or hamper parasitoid counteradaptations, to determine exactly how insects use symbionts in their defence against natural enemies. AUTHOR CONTRIBUTIONS L.M.H. conceived the idea, acquired funding, led the investigation and supervision of the team. T. W. and A.A.R. conducted field collections, and formal analysis of the data. T.F. conducted additional analyses. All authors contributed to the drafting, reviewing, and editing of the manuscript. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. CONFLICT OF INTERESTS We the authors declare no conflicting interests. DATA ACCESSIBILITY Data and scripts are available online: ########. The GenBank accession numbers for Sanger sequences determined in this study are ######## to ######## ( Hamiltonella MLST). The GenBank accession numbers for Illumina sequences determined in this study are ######## to ######## (Aphid COI), and ######## – ######## (Parasitoid COI). The GenBank accession number for the raw data of Illumina sequencing used in this study is ##########. ACKNOLWEDGEMENTS This work was funded by Leverhulme grant (RPG-2020-211) awarded to L.M.H. REFERENCES 1. ↵ Ahmed , M.Z. , Li , S.J. , Xue , X. , Yin , X.J. , Ren , S.X. , Jiggins , F.M. , et al. ( 2015 ). The intracellular bacterium Wolbachia uses parasitoid wasps as phoretic vectors for efficient horizontal transmission . PLoS Pathogens , 11 , e1004672 . OpenUrl CrossRef 2. ↵ Ahmed , Q. , Agarwal , M. , Alobaidi , R. , Zhang , H. & Ren , Y . ( 2022 ). Response of aphid parasitoids to volatile organic compounds from undamaged and infested Brassica oleracea with Myzus persicae . Molecules , 27 , 1522 . OpenUrl 3. ↵ Ali , M.Y. , Naseem , T. , Zhang , J. , Pan , M. , Zhang , F. & Liu , T.X . ( 2022 ). Plant volatiles and herbivore induced plant volatiles from chili pepper act as attractant of the aphid parasitoid Aphelinus varipes (Hymenoptera: Aphelinidae) . Plants , 11 , 1350 . OpenUrl 4. ↵ Bates , D. , Mächler , M. , Bolker , B. & Walker , S . ( 2015 ). Fitting linear mixed-effects models using lme4 . Journal of Statistical Software , 67 , 1 – 48 . OpenUrl 5. ↵ Blüthgen , N. , Menzel , F. , Hovestadt , T. , Fiala , B. , & Blüthgen , N . ( 2007 ). Specialization, constraints, and conflicting interests in mutualistic networks . Current Biology , 17 , 341 – 346 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Callahan , B.J. , McMurdie , P.J. , Rosen , M.J. , Han , A.W. , Johnson , A.J.A. & Holmes , S.P . ( 2016 ). DADA2: high-resolution sample inference from Illumina amplicon data . Nature Methods , 13 , 581 – 583 . OpenUrl 7. ↵ Carpenter , M. , Peng , L. , Smith , A.H. , Joffe , J. , O’Connor , M. , Oliver , K.M. , et al. ( 2021 ). Frequent drivers, occasional passengers: signals of symbiont-driven seasonal adaptation and hitchhiking in the pea aphid, Acyrthosiphon pisum . Insects , 12 , 805 . OpenUrl 8. ↵ Caspi-Fluger , A. , Inbar , M. , Mozes-Daube , N. , Katzir , N. , Portnoy , V. , Belausov , E. , et al. ( 2012 ). Horizontal transmission of the insect symbiont Rickettsia is plant-mediated . Proceedings of the Royal Society B: Biological Sciences , 279 , 1791 – 1796 . OpenUrl CrossRef PubMed 9. ↵ Cayetano , L. , Rothacher , L. , Simon , J.C. & Vorburger , C . ( 2015 ). Cheaper is not always worse: strongly protective isolates of a defensive symbiont are less costly to the aphid host . Proceedings of the Royal Society B: Biological Sciences , 282 , 20142333 . OpenUrl CrossRef PubMed 10. ↵ Chrostek , E. , Pelz-Stelinski , K. , Hurst , G.D.D. & Hughes , G.L . ( 2017 ). Horizontal transmission of intracellular insect symbionts via plants . Frontiers in Microbiology , 8 , 2237 . OpenUrl 11. ↵ Cornwallis , C.K. , van ‘t Padje , A. , Ellers , J. , Klein , M. , Jackson , R. , Kiers , E.T. , et al. ( 2023 ). Symbioses shape feeding niches and diversification across insects . Nature Ecology & Evolution , 7 , 1022 – 1044 . OpenUrl 12. ↵ Csárdi , G. , Nepusz , T. , Traag , V. , Horvát , S. , Zanini , F. , Noom , D. , et al. ( 2024 ). igraph: Network analysis and visualization in R . Available from: http://igraph.org/ (accessed 18 July 2024). 13. ↵ Degnan , P.H. & Moran , N.A . ( 2008 ). Evolutionary genetics of a defensive facultative symbiont of insects: exchange of toxin-encoding bacteriophage . Molecular Ecology , 17 , 916 – 929 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Dion , E. , Zélé , F. , Simon , J.C. & Outreman , Y . ( 2011 ). Rapid evolution of parasitoids when faced with the symbiont-mediated resistance of their hosts . Journal of Evolutionary Biology , 24 , 741 – 750 . OpenUrl CrossRef PubMed 15. ↵ Dormann , C.F. , Gruber , B. & Fruend , J . ( 2008 ). Introducing the bipartite package: analysing ecological networks . R News , 8 , 8 – 11 . OpenUrl 16. ↵ Dykstra , H.R. , Weldon , S.R. , Martinez , A.J. , White , J.A. , Hopper , K.R. , Heimpel , G.E. , et al. ( 2014 ). Factors limiting the spread of the protective symbiont Hamiltonella defensa in Aphis craccivora aphids . Applied Environmental Microbiology , 80 , 5818 – 5827 . OpenUrl Abstract / FREE Full Text 17. ↵ Ferrari , J. , Darby , A.C. , Daniell , T.J. , Godfray , H.C.J. & Douglas , A.E . ( 2004 ). Linking the bacterial community in pea aphids with host-plant use and natural enemy resistance . Ecological Entomology , 29 , 60 – 65 . OpenUrl 18. ↵ Ferrari , J. , West , J.A. , Via , S. & Godfray , H.C.J . ( 2012 ). Population genetic structure and secondary symbionts in host-associated populations of the pea aphid complex . Evolution , 66 , 375 – 390 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Frago , E. , Mala , M. , Weldegergis , B.T. , Yang , C. , McLean , A. , Godfray , H.C.J. , et al. ( 2017 ). Symbionts protect aphids from parasitic wasps by attenuating herbivore-induced plant volatiles . Nature Communications , 8 , 1860 . OpenUrl 20. ↵ Gehrer , L. & Vorburger , C . ( 2012 ). Parasitoids as vectors of facultative bacterial endosymbionts in aphids . Biology Letters , 8 , 613 – 615 . OpenUrl 21. ↵ Gimmi , E. & Vorburger , C . ( 2021 ). Strong genotype-by-genotype interactions between aphid-defensive symbionts and parasitoids persist across different biotic environments . Journal of Evolutionary Biology , 34 , 1944 – 1953 . OpenUrl CrossRef 22. ↵ Gimmi , E. & Vorburger , C . ( 2024 ). High specificity of symbiont-conferred resistance in an aphid-parasitoid field community . Journal of Evolutionary Biology , 37 , 162 – 170 . OpenUrl 23. ↵ Gimmi , E. , Wallisch , J. & Vorburger , C . ( 2024 ). Ecological divergence despite common mating sites: genotypes and symbiotypes shed light on cryptic diversity in the black bean aphid species complex . Heredity , 132 , 320 – 330 . OpenUrl 24. ↵ Goldstein , E.B. , de Anda Acosta , Y. , Henry , L.M. & Parker , B.J. ( 2023 ) Variation in density, immune gene suppression, and coinfection outcomes among strains of the aphid endosymbiont Regiella insecticola . Evolution , 77 , 1704 -1711. OpenUrl 25. ↵ Gonella , E. , Pajoro , M. , Marzorati , M. , Crotti , E. , Mandrioli , M. , Pontini , M. , et al. ( 2015 ). Plant-mediated interspecific horizontal transmission of an intracellular symbiont in insects . Scientific Reports , 5 , 15811 . OpenUrl 26. ↵ Guindon , S. , Dufayard , J.F. , Lefort , V. , Anisimova , M. , Hordijk , W. & Gascuel , O . ( 2010 ). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0 . Systematic Biology , 59 , 307 – 321 . OpenUrl CrossRef PubMed Web of Science 27. ↵ Hafer-Hahmann , N. & Vorburger , C. ( 2020 ). Parasitoids as drivers of symbiont diversity in an insect host . Ecology Letters , 23 , 1232 – 1241 . OpenUrl 28. ↵ Hafer-Hahmann , N. & Vorburger , C. ( 2024 ). Parasitoid species diversity has no effect on protective symbiont diversity in experimental host-parasitoid populations . Ecology and Evolution , 14 , e11090 . OpenUrl 29. ↵ Henry , L.M. , Peccoud , J. , Simon , J.C. , Hadfield , J.D. , Maiden , M.J.C. , Ferrari , J. , et al. ( 2013 ). Horizontally transmitted symbionts and host colonization of ecological niches . Current Biology , 23 , 1713 – 1717 . OpenUrl CrossRef PubMed 30. ↵ Henry , L.M. , Maiden , M.C.J. , Ferrari , J. & Godfray , H.C.J . ( 2015 ). Insect life history and the evolution of bacterial mutualism . Ecology Letters , 18 , 516 – 525 . OpenUrl CrossRef PubMed 31. ↵ Henry , Y. , Brechbühler , E. & Vorburger , C . ( 2022 ). Gated communities: inter– and intraspecific diversity of endosymbionts across four sympatric aphid species . Frontiers in Ecology and Evolution , 10 , 816184 . OpenUrl 32. ↵ Heyworth , E.R. , Smee , M.R. & Ferrari , J . ( 2020 ). Aphid facultative symbionts aid recovery of their obligate symbiont and their host after heat stress . Frontiers in Ecology and Evolution , 8 , 56 . OpenUrl 33. ↵ Higashi , C.H.V. , Nichols , W.L. , Chevignon , G. , Patel , V. , Allison , S.E. , Kim , K.L. , et al. ( 2023 ). An aphid symbiont confers protection against a specialized RNA virus, another increases vulnerability to the same pathogen . Molecular Ecology , 32 , 936 – 950 . OpenUrl 34. ↵ Jackson , R. , Monnin , D. , Patapiou , P.A. , Golding , G. , Helanterä , H. , Oettler , J. , et al. ( 2022 ). Convergent evolution of a labile nutritional symbiosis in ants . The ISME Journal , 16 , 2114 – 2122 . OpenUrl 35. ↵ Jackson , R. , Parapiou , P.A. , Golding , G. , Helanterä , H. , Economou , C.K. , Chapuisat , M. , Henry , L.M . ( 2023 ) Evidence of phylosymbiosis in Formica ants . Frontiers in Microbiology , 14 , 1044286 . OpenUrl 36. ↵ Jaenike , J . ( 2012 ). Population genetics of beneficial heritable symbionts . Trends in Ecology and Evolution , 27 , 226 – 232 . OpenUrl 37. ↵ Kaech , H. & Vorburger , C . ( 2021 ). Horizontal transmission of the heritable protective endosymbiont Hamiltonella defensa depends on titre and haplotype . Frontiers in Microbiology , 11 , 628755 . OpenUrl 38. ↵ Leclair , M. , Buchard , C. , Mahéo , F. , Simon , J.C. & Outreman , Y . ( 2021 ). A link between communities of protective endosymbionts and parasitoids of the pea aphid revealed in unmanipulated agricultural systems . Frontiers in Ecology and Evolution , 9 , 618331 . OpenUrl 39. ↵ Li , Q. , Fan , J. , Sun , J. , Wang , M.Q. & Chen , J . ( 2018 ). Plant-mediated horizontal transmission of Hamiltonella defensa in the wheat aphid Sitobion miscanthi . Journal of Agricultural and Food Chemistry , 66 , 13367 – 13377 . OpenUrl 40. ↵ Li , S.J. , Ahmed , M.Z. , Lv , N. , Shi , P.Q. , Wang , X.M. , Huang , J.L. , et al. ( 2017 ). Plant–mediated horizontal transmission of Wolbachia between whiteflies . The ISME Journal , 11 , 1019 – 1028 . OpenUrl 41. ↵ Łukasik , P. , van Asch , M. , Guo , H. , Ferrari , J. & Charles J. , Godfray , H.C.J ( 2013 ). Unrelated facultative endosymbionts protect aphids against a fungal pathogen . Ecology Letters , 16 , 214 – 218 . OpenUrl CrossRef PubMed 42. ↵ Łukasik , P. , Guo , H. , van Asch , M. , Henry , L.M. , Godfray , H.C.J. & Ferrari , J. ( 2015 ). Horizontal transfer of facultative endosymbionts is limited by host relatedness . Evolution , 69 , 2757 – 2766 . OpenUrl CrossRef PubMed 43. ↵ Lynn-Bell , N.L. , Strand , M.R. & Oliver , K.M . ( 2019 ). Bacteriophage acquisition restores protective mutualism . Microbiology , 165 , 985 – 989 . OpenUrl 44. Mantel , N . ( 1967 ). The detection of disease clustering and a generalized regression approach . Cancer Research , 27 , 209 – 20 . OpenUrl Abstract / FREE Full Text 45. ↵ Martinez , A.J. , Kim , K.L. , Harmon , J.P. & Oliver , K.M . ( 2016 ). Specificity of multi-modal aphid defenses against two rival parasitoids . PLoS One , 11 , e0154670 . OpenUrl CrossRef 46. ↵ Mateos , M. , Winter , L. , Winter , C. , Higareda-Alvear , V.M. , Martinez-Romero , E. & Xie , J. ( 2016 ). Independent origins of resistance or susceptibility of parasitic wasps to a defensive symbiont . Ecology and Evolution , 6 , 2679 – 2687 . OpenUrl 47. ↵ McLean , A.H.C. & Godfray , H.C.J . ( 2015 ). Evidence for specificity in symbiont-conferred protection against parasitoids . Proceedings of the Royal Society B: Biological Sciences , 282 , 20150977 . OpenUrl CrossRef PubMed 48. ↵ McLean , A.H.C. & Godfray , H.C.J . ( 2017 ). The outcome of competition between two parasitoid species is influenced by a facultative symbiont of their aphid host . Functional Ecology , 31 , 927 – 933 . OpenUrl 49. ↵ McLean , A.H.C. , Godfray , H.C.J. , Ellers , J. & Henry , L.M . ( 2019 ). Host relatedness influences the composition of aphid microbiomes . Environmental Microbiology Reports , 11 , 808 – 816 . OpenUrl CrossRef 50. ↵ Niepoth , N. , Ellers , J. & Henry , L.M . ( 2018 ). Symbiont interactions with non-native hosts limit the formation of new symbioses . BMC Evolutionary Biology , 18 , 27 . OpenUrl 51. Oksanen , J. , Simpson , G.L. , Blanchet , F.G. , Kindt , R. , Legendre , P. , Minchin , P.R. , et al. ( 2022 ). vegan: Community ecology package . Available from: https://CRAN.R-project.org/package=vegan/ (accessed 18 July 2024). 52. ↵ Oliver , K.M. , Russell , J.A. , Moran , N.A. & Hunter , M.S . ( 2003 ). Facultative bacterial symbionts in aphids confer resistance to parasitic wasps . Proceedings of the National Academy of Sciences , 100 , 1803 – 1807 . OpenUrl Abstract / FREE Full Text 53. ↵ Oliver , K.M. , Campos , J. , Moran , N.A. & Hunter , M.S . ( 2008 ). Population dynamics of defensive symbionts in aphids . Proceedings of the Royal Society B: Biological Sciences , 275 , 293 – 299 . OpenUrl CrossRef PubMed Web of Science 54. ↵ Oliver , K.M. & Higashi , C.H . ( 2019 ). Variations on a protective theme: Hamiltonella defensa infections in aphids variably impact parasitoid success . Current Opinion in Insect Science , 32 , 1 – 7 . OpenUrl 55. ↵ Oliver , K.M. , & Perlman , S.J . ( 2020 ) Toxin-mediated protection against natural enemies by insect defensive symbionts . Advances in insect physiology , 58 , 277 – 316 . OpenUrl 56. ↵ Osaka , R. , Watada , M. , Kageyama , D. & Nomura , M . ( 2010 ). Population dynamics of a maternally-transmitted Spiroplasma infection in Drosophila hydei . Symbiosis , 52 , 41 – 45 . OpenUrl 57. ↵ Paradis , E. & Schliep , K . ( 2019 ). ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R . Bioinformatics , 35 , 526 – 528 . OpenUrl CrossRef PubMed 58. ↵ Pons , I. , Renoz , F. , Noël , C. & Hance , T . ( 2019 ). Circulation of the cultivable symbiont Serratia symbiotica in aphids is mediated by plants . Frontiers in Microbiology , 10 , 764 . OpenUrl 59. R Core Team . ( 2023 ). R: A language and environment for statistical computing . Available from https://www.r-project.org/ (accessed 18 July 2024). 60. ↵ Rouchet , R. & Vorburger , C . ( 2012 ). Strong specificity in the interaction between parasitoids and symbiont-protected hosts . Journal of Evolutionary Biology , 25 , 2369 – 2375 . OpenUrl CrossRef PubMed 61. ↵ Russell , J.A. , Latorre , A. , Sabater-Muñoz , B. , Moya , A. & Moran , N.A. ( 2003 ). Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea . Molecular Ecology , 12 , 1061 – 1075 . OpenUrl CrossRef PubMed Web of Science 62. ↵ Smith , A.H. , O’Connor , M.P. , Deal , B. , Kotzer , C. , Lee , A. , Wagner , B. , et al. ( 2021 ). Does getting defensive get you anywhere? — Seasonal balancing selection, temperature, and parasitoids shape real-world, protective endosymbiont dynamics in the pea aphid . Molecular Ecology , 30 , 2449 – 2472 . OpenUrl CrossRef 63. ↵ Soleimannejad , S. , Ross , P.A. & Hoffmann , A.A . ( 2023 ). Effect of Rickettsiella viridis endosymbionts introduced into Myzus persicae aphids on parasitism by Diaeretiella rapae : a combined strategy for aphid control? Biological Control , 187 , 105377 . OpenUrl 64. ↵ Tamura , K. , Stecher , G. & Kumar , S . ( 2021 ). MEGA11: Molecular evolutionary genetics analysis version 11 . Molecular Biology and Evolution , 38 , 3022 – 3027 . OpenUrl CrossRef 65. ↵ Toju , H. & Fukatsu , T . ( 2011 ). Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants . Molecular Ecology , 20 , 853 – 868 . OpenUrl CrossRef PubMed Web of Science 66. ↵ Tougeron , K. & Iltis , C . ( 2022 ). Impact of heat stress on the fitness outcomes of symbiotic infection in aphids: a meta-analysis . Proceedings of the Royal Society B: Biological Sciences , 289 , 20212660 . OpenUrl 67. ↵ Tsuchida , T. , Koga , R. , Shibao , H. , Matsumoto , T. & Fukatsu , T . ( 2002 ). Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid, Acyrthosiphon pisum . Molecular Ecology , 11 , 2123 – 2135 . OpenUrl CrossRef PubMed Web of Science 68. ↵ Tzuri , N. , Caspi-Fluger , A. , Betelman , K. , Rohkin Shalom , S. & Chiel , E . ( 2021 ). Horizontal transmission of microbial symbionts within a guild of fly parasitoids . Microbial Ecology , 81 , 818 – 827 . OpenUrl 69. ↵ Vorburger , C. , Gehrer , L. & Rodriguez , P . ( 2010 ). A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids . Biology Letters , 6 , 109 – 111 . OpenUrl 70. ↵ Vorburger , C . ( 2022 ). Defensive symbionts and the evolution of parasitoid host specialization . Annual Review of Entomology , 67 , 329 – 346 . OpenUrl 71. ↵ Weldon , S.R. , Russell , J.A. & Oliver , K.M . ( 2020 ). More is not always better: coinfections with defensive symbionts generate highly variable outcomes . Applied Environmental Microbiology , 86 , e02537 – 19 . OpenUrl CrossRef 72. ↵ Wickham , H . ( 2016 ). ggplot2: Elegant graphics for data analysis . Springer-Verlag, New York . 73. Wickham , H. , François , R. , Henry , L. , Müller , K. & Vaughan , D. ( 2023 ). dplyr: A grammar of data manipulation . Available from: https://CRAN.R-project.org/package=dplyr/ (accessed 18 July 2024). 74. ↵ Wu , T. , Monnin , D. , Lee , R.A.R. & Henry , L.M . ( 2022 ). Local adaptation to hosts and parasitoids shape Hamiltonella defensa genotypes across aphid species . Proceedings of the Royal Society B: Biological Sciences , 289 , 20221269 . OpenUrl CrossRef 75. ↵ Xie , J. , Butler , S. , Sanchez , G. & Mateos , M . ( 2014 ). Male killing Spiroplasma protects Drosophila melanogaster against two parasitoid wasps . Heredity , 112 , 399 – 408 . OpenUrl CrossRef PubMed 76. ↵ Zhao , D. , Zhang , Z. , Niu , H. & Guo , H . ( 2023 ). Pathogens are an important driving force for the rapid spread of symbionts in an insect host . Nature Ecology & Evolution , 7 , 1667 – 1681 . OpenUrl View the discussion thread. Back to top Previous Next Posted July 24, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Defensive symbiont genotype distributions are linked to parasitoid attack networks Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Defensive symbiont genotype distributions are linked to parasitoid attack networks Taoping Wu , Anoushka A. Rodrigues , Tom Fayle , Lee M. Henry bioRxiv 2024.07.24.604610; doi: https://doi.org/10.1101/2024.07.24.604610 Share This Article: Copy Citation Tools Defensive symbiont genotype distributions are linked to parasitoid attack networks Taoping Wu , Anoushka A. Rodrigues , Tom Fayle , Lee M. Henry bioRxiv 2024.07.24.604610; doi: https://doi.org/10.1101/2024.07.24.604610 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Ecology Subject Areas All Articles Animal Behavior and Cognition (7652) Biochemistry (17758) Bioengineering (13944) Bioinformatics (42096) Biophysics (21508) Cancer Biology (18666) Cell Biology (25588) Clinical Trials (138) Developmental Biology (13412) Ecology (19956) Epidemiology (2067) Evolutionary Biology (24390) Genetics (15643) Genomics (22578) Immunology (17788) Microbiology (40522) Molecular Biology (17222) Neuroscience (88845) Paleontology (667) Pathology (2847) Pharmacology and Toxicology (4841) Physiology (7668) Plant Biology (15182) Scientific Communication and Education (2048) Synthetic Biology (4312) Systems Biology (9842) Zoology (2274)