Are competitive interactions between aphid clones mediated by facultative endosymbionts?

Are competitive interactions between aphid clones mediated by facultative endosymbionts? | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Are competitive interactions between aphid clones mediated by facultative endosymbionts? Mario G. Moya-Hernández, María E. Rubio-Meléndez, Francisca A. Zepeda-Paulo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4021194/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacterial endosymbionts are key components of aphid biology, as they modify several traits of their insect hosts. Here we studied how bacterial facultative endosymbionts affect the competitive interactions between aphid clones. To address this, we studied intraclonal and interclonal interactions between the two most common clones (G1 and G2) of the cereal aphid Sitobion avenae (Fabricius), including the role of the facultative endosymbiont Regiella insecticola in the outcome of these interactions in a shared host (wheat). The results of this study reveal significant variability in the population growth rates of aphid clones under competitive and non-competitive environments. That trade-off in competitive interactions among aphid clones can influence the dynamics of aphid populations and impact on plant growth and structure. While facultative endosymbionts like R. insecticola do not play a significant role in directly mediating the competitive interactions of aphid clones or affecting specific plant traits, their presence does enhance aphid performance. Aphids harbouring R. insecticola showed higher growth rates in various coexistence scenarios and across different clones with a concurrently reduced capacity to damage host plants, which suggests that R. insecticola produces ecologically relevant consequences for aphids in cereal fields. interclonal intraclonal interactions performance winged morph aphid endosymbionts Regiella insecticola Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Animal symbionts can play a decisive role in determining the outcome of ecological and evolutionary processes (Moran 2006 ; McFall-Ngaia et al. 2013; Vorburger and Perlman 2018 ). Furthermore, symbiosis has allowed many animal species to acquire novel functions, occupy new ecological niches, and influence their ecological interactions with other organisms (Oliver et al. 2003 ; Scarborough et al. 2005 ; Sudakaran et al. 2017 ). While the impact of symbiosis has been mostly studied in mutualistic and predatory ecological interactions (Douglas 2009 ; Feldhaar 2011 ), little is known about its role in competitive interactions. Aphids are phytophagous insects with worldwide distribution, representing an important agricultural pest of several crops (Leight and Emden 2017). Bacterial endosymbionts have been shown to play a key role in aphid biology (Oliver et al. 2010 ). Aphids are hosts of a primary endosymbiont, Buchnera aphidicola , a bacterium that is vertically transmitted and synthesizes essential amino acids and other nutrients, which are necessary for aphid reproduction and survival (Koga et al. 2003 ). Moreover, common associations between aphids and several secondary or facultative bacterial endosymbionts have been evidenced in different aphid populations. Facultative endosymbionts are predominantly vertically transmitted from females to offspring but also horizontal transfer occasionally could occur within and between species (Ferrari and Vavre 2011 ; Oliver et al. 2010 ). Harbouring endosymbionts may directly influence host biology by conferring conditional benefits and/or fitness costs on hosts(Ferrari and Vavre 2011 ; Pons et al. 2019 a; Kaech et al. 2022 ). Among the most well-known endosymbiont-mediated effects on aphids is the protection against parasitoids, such when the pea aphid ( Acyrthosiphon pisum ) is infected with the facultative endosymbiont Hamiltonella defense (Oliver et al. 2003 ). Moreover, the facultative endosymbiont Regiella insecticola protects to the pea aphid A. pisum from the lethal fungus Pandora neoaphidis (Scarborough et al. 2005 ). According to Tsuchida et al., ( 2004 ), pea aphids harbour R. insecticola improves significantly its performance on clover ( Trifolium sp.). Besides, facultative endosymbionts can indirectly shape ecological interactions by altering traits that influence the mutualistic and antagonist relationships of aphids. For instance, endosymbionts can change the honeydew and cuticular composition of aphids, which can affect interspecific communication with their mutualistic ants (Schillewaert et al. 2017 ; Hertaeg et al. 2021 ). Furthermore, the predatory activity of ladybirds on aphids can be altered by harboring facultative endosymbionts, either positively or negatively (Ramírez-Cáceres et al. 2019 ; Luo et al. 2022 ). On the other hand, facultative endosymbiont can influence the trophic relationships such as aphid-host plant interactions. In this sense, Wagner et al. ( 2015 ) showed that despite the low clonal variability in the aphid Aphis craccivora , the presence or absence of maternal inhered facultative endosymbionts can act as facilitators for the expansion or restriction of aphid clones on new plant hosts. In addition, endosymbionts can be horizontally transferred between aphids and host plants, entering plants, and modify the functioning of the plant by promote the root growth (larger Root: shoot) (Pons et al. 2019b ). Thereby, aphid clone-facultative endosymbiont associations could be modulating the relative success and the ecological interactions of aphid clones on their host plants. Although facultative endosymbiont-mediated effects on aphids have been widely studied in several aphid species, little is known about the role of facultative endosymbionts on the competitive interactions among aphid clones. Due to its predominant asexual reproduction (parthenogenetic), aphid populations are commonly conformed by a clonal genotype set subjected to diverse ecological stressors and seasonal changes in their environment. Furthermore, variability and rapid seasonal shifts in the frequency of endosymbiont infection have been reported among aphid clones within-populations (Zepeda-Paulo et al. 2017 , 2021). Interclonal interactions could play an important role in the understating of mechanisms that facilitate the coexistence of clones within aphid pest species (Bruijninget al. 2019 ), however the effect of facultative endosymbionts on the competitive interactions of aphids has been little aimed. For example, Turcotte et al. ( 2011a ) evaluated the population dynamics of three clones of the green peach aphid ( M. persicae s. str. ), observing that two of the three clones studied increased their population growth rate between 28–34% under interclonal competition compared to the control treatment (without interclonal competition). A similar study carried out with those clones in the field showed an increase in the population growth rate of 47% and a 67% increase in the population under interclonal competition (Turcotte et al. 2011b ). These studies suggest that interclonal interactions could be common within aphid populations, however to our knowledge there is no published evidence on how facultative endosymbionts affect the inter and intra-clonal interactions in the aphid group, such as when different clones coexist in the same host plant. In Chile, the populations of Sitobion avenae exhibit a low genetic variability, characterized by a few predominant clones that are able to successfully colonize different cultivated and wild host plants through different climatic zones, which have been maintained over time (“superclones”) (Figueroa et al. 2005 ). The success of these abundant and persistent clones in the field is likely affected by specific associations of aphid clones and endosymbionts found in the field (Zepeda-Paulo et al. 2021). Also, in this aphid species has been discarded a protection-mediated role by facultative endosymbionts ( H. defensa and R. insecticola ) against parasitoids (Łukasik et al. 2013 ; Zepeda-Paulo et al. 2017 ). Given that the presence of facultative endosymbionts usually generates positive effects on the individuals that host them, our working hypothesis was that the coexistence of S. avenae clones is modulated by the presence of the facultative endosymbiont R. insecticola . Thus, the aim of this study was to analyse the biological effects of facultative endosymbionts on the ecological interactions of aphid clones to answer the following question: what is the impact of facultative endosymbiosis on the intra and interclonal competitive interactions of aphid clones? and to what extent does the outcome of competitive interactions depend on the genotypic composition of the interacting clones? These questions were addressed by subjecting the two prevalent clones of the aphid S. avenae , both infected and uninfected with the most common facultative endosymbiont R. insecticola , rearing under competition on a common host plant and evaluates aphid population growth rates and life-history traits, including the consequences on functional traits of the host plants (root/shoot ratio). 2. MATERIALS AND METHODS 2.1 Aphid sampling and rearing conditions Two genotypes of the aphid S. avenae were established from field as clonal lineages and assayed in the present study. Multilocus genotypes were determined by amplifying eight microsatellite loci described in S. avenae (Table 1 ). The two aphid genotypes (clone G1 and G2) hereafter used in this study corresponded to individuals collected on wheat fields with a widespread distribution and frequency across different agroclimatic zones sampled (Zepeda-Paulo et al., 2017 ). Table 1 Endosymbiont status and multilocus genotype of clones of the cereal aphid Sitobion avenae based on eight microsatellite loci used in the coexistence experiments. Clone Code Endosymbiont status Sm11 S3.43 S16b S30 S4Σ S5L Sm17 Sm10 +G1 Regiella insecticola 160164 203203 224226 177179 175181 243245 115115 185187 -G1 Disinfected 160164 203203 224226 177179 175181 243245 115115 185187 +G2 Regiella insecticola 160164 203212 205205 177179 181185 241245 115115 185185 -G2 Naturally uninfected 160164 203212 205205 177179 181185 241245 115115 185185 Alive specimens were transferred to the laboratory and composed separately clonal lineages. The colony of clone G1 was collected naturally infected with R. insecticola , was split in two parts, one part was disinfected with antibiotics following the procedure described by Koga et al. ( 2007 ) and Simon et al. ( 2011 ), and the other part was kept as collected. This allowed the rearing of G1 with and without the presence of R. insecticola (hereafter + G1 and –G1, respectively) (Table 1 ). Differently, the aphid genotype G2 was collected both clones naturally harbouring and uninfected the R. insecticola (hereafter + G2 and –G2, respectively) (Table 1 ). All aphid clones, infected and uninfected with R. insecticola , were independently maintained in the laboratory on potted plants of wheat ( Triticum aestivum ; cv. Pantera-INIA) in rearing cages (50 x 40 x 30 cm) under controlled climatic conditions at a temperature of 20 ± 2 ° C and 16L: 8D photoperiod and watered every three days. 2.2 Genotyping and endosymbiont detected in aphid samples Genomic DNA was extracted from each aphid using the “salting-out” method (Sunnucks and Hales 1996 ). Each aphid was genotyped with eight discriminant microsatellite loci (Table 1 ). These microsatellites have been previously isolated from S. avenae by Wilson et al. ( 2004 ) and extensively used to determine the genetic diversity worldwide (Simon et al., 1999 ; Llewellyn et al., 2004 ; Figueroa et al. 2005 ; Xin et al. 2014 ). The microsatellite loci were amplified modifying the forward primer with M13(-21) tail at the 5’ end for each microsatellite. The reaction was performed adding the sequence-specific forward primer with M13(-21) tail at its 5′ end, the sequence-specific reverse primer and the universal FAM or VIC or NED fluorescent-labeled M13(-21) primer (Schuelke, 2000 ). The PCR products were genotyped in Macrogen Inc (Rep. of Korea) and the electropherograms obtained were analysed using the in GENMARK version 1.3 (Borodovsky and McIninch 1993 ). In other hand, the presence of secondary endosymbionts was determined by molecular detection in each aphid. Using a PCR multiplex (Peccoud et al. 2014 ) for eight facultative endosymbionts in each clone, for all treatment. In this sense, first standardizing simplex methods PCR for all loci, using the obligatory bacterial endosymbiont of aphids, Buchnera aphidicola , as positive control for PCR reaction. Also, in multiplex PCR were used samples of the aphid Acyrthosiphon pisum (with known endosymbiont infections) as positive control in each amplification diagnostic. Polymerase chain reaction (PCR) amplifications were carried out in an Viriti Thermal Cycler (Applied Biosystems), under the following conditions: 5 min of denaturation at 94°C followed by 30 cycles with 30 s of denaturation at 94°C, 30 s at 58°C of annealing temperature and 1 min of elongation at 72°C and a ended with 5 min elongation at 72°C. Sequences were visualized through electrophoresis agarose gel (1.5%), using a GeneRuler 100 bp Plus DNA Ladder (Fermentas) for determining the size of each sequence. Each facultative endosymbiont was identified according to the size of the sequence in base pairs (bp) obtained from the total DNA of each individual aphid (Peccoud et al., 2014 ). The following diagnostic sizes were used: Spiroplasma (1500 bp), Serratia symbiotica (1140 bp), Regiella insecticola (840 bp), Rickettsia (600 bp), Hamiltonella defensa (480 bp), Fukatsuia symbiotica (500 bp) and Rickettsiella sp (300 bp) and Buchnera aphidicola (270 bp) (Peccoud et al., 2014 ). All detections of endosymbionts were positive for Buchnera aphidicola , in this sense the quality of DNA was enough. For the case of the detection in clones + G1 and + G2 we only detected R. insecticola and no other secondary endosymbionts was reported for our colonies of S. avenae for all treatment. 2.3 Experiments design To study the influence of facultative endosymbionts on the reproduction and competition of aphid clones, we performed a 2 x 2 x 2 experimental design to examinate the population growth of two aphid genotypes (G1 and G2) with [+] and without [-] the presence of the endosymbiont R. insecticola exposed to two coexistence treatments; i) single aphid clone (+ G1, -G1, +G2 and -G2), and ii) a clonal mixing (G1-G1, +G2-G2, + G1 + G2, +G1-G2, -G1 + G2 and -G1-G2). The factors studied were genotype (G1 and G2), endosymbiont (with [+] and without [-] the presence of R. insecticola ) and coexistence status (single and coexisting genotypes). The factors were combined originating ten treatments with twenty replicates either one (Table 2 ). Each replicate was composed of a microcosm, which included three wheat seedlings grow in a pot (650 cc) containing a substrate composed by 1:1 (vermiculite: perlite). Each pot was protected using a transparent breezy plastic tube with nylon mesh-covered openings. All treatments the aphid colonies started with age-synchronized aphids. For this, 50 individuals of one genotype of approximately the same age were taken from the rearing cages and placed on wheat. After 24 hours, all adult aphids were removed, and only new-born nymphs were kept. The individuals starting the colonies in all the treatments were taken from this stock of age-synchronized nymphs. In treatments composed by solitary colonies (+ G1, -G1, +G2 and -G2), four nymphs were placed on the wheat seedlings in each microcosm. In treatments of coexisting aphids, two nymphs per genotype /infection status were used (G1-G1, +G2-G2, + G1 + G2, +G1-G2, -G1 + G2 and -G1-G2). This allowed the use of a similar density of aphids per microcosm in all ten treatments. Aphid numbers were counted by visual inspection at 0, 7, 15 and 26 days in the different treatments, similarly to reported by Turcotte et al. ( 2011b ). At day 26, all aphids were removed and place on a white tray with alcohol 95% for a detailed counting, including all nymphs, winged and wingless adults. In the case of coexisting colonies, because aphids of different genotypes or harbouring R. insecticola were not able to be distinguished phenotypically, individuals were genotyped by microsatellite loci and endosymbiont PCR-detected. Due to the large number of specimens recovered at the end of the experiment, the microsatellite genotyping and PCR-based endosymbiont detection was performed to a sub-sampled containing the 50% of the adults (wingless and winged aphids) obtained from each pot at the end experiment (day 26). Table 2 Experimental design: the coexistence treatments are termed according to the presence of the aphid genotypes (G1 and G2) with (+) and without (-) the presence of the facultative endosymbiont R. insecticola . Treatments Coexistence Number of replicates +G1 Control treatment 20 -G1 Control treatment 20 +G2 Control treatment 20 -G2 Control treatment 20 +G1-G1 Intraclonal 20 +G2-G2 Intraclonal 20 +G1 + G2 Interclonal 20 +G1-G2 Interclonal 20 -G1 + G2 Interclonal 20 -G1-G2 Interclonal 20 2.4 Population growth rate (PGR) and population dynamic The population growth rate (PGR) in the different treatments was estimated as ln (Nf + 0.001) - ln (Ni) / (tf – ti), where Nf and Ni refers the final and initial number of aphids, respectively, and (tf – ti) is the number of days of the experiment (26 days) (Thomas et al. 2011 ). The 0.001 was added to Nf because in some replicates final numbers were zero, as recommended by Thomas et al. ( 2011 ). The initial population size was 4 individuals in all replicates. In addition, the total number of aphids was studied at days 0, 7, 15 and 26 in the different treatments allowing the assessment of the temporal variation in the abundance of aphids. 2.5 Fresh weight and proportion of winged aphids To evaluate whether the facultative endosymbiont R. insecticola influenced aphid body mass, as described in other aphid systems (Oliver et al. 2008 ), 30 adult individuals of each genotype harbouring or not harbouring R. insecticola were carefully identified and selected using a Zeiss Stemi DV4stereomicroscope, and then the weight was measured using a Mettler Toledo XS3DU microbalance. The proportion of winged aphid was calculated by dividing the density of winged individuals by the total aphid density (Petermann et al., 2010 ). 2.6 Relative root/shoot of plants The root/shoot ratio of wheat seedling used for feeding aphids in the 10 treatments was calculated as the ratio of total root dry mass to total leaf dry mass. Once the experiment finished at day 26, each plant was dried at 60°C for 5 days and weighed and dry weight was recorded. An additional treatment composed by 20 control plants was also implemented. The relative change in the plant root/shoot ratio of each treatment was obtained by calculating the coefficient between the root/shoot ratio of each treatment and control plants using a microbalance Radwag. 2.7 Data analysis PGR data were transformed to antilog (exp) and compared using a generalized linear model (GLM) with a Gaussian distribution. PGR data analysis was performed on the experimental design contemplating 2 (genotypes G1 and G2) x 2 (with and without R. insecticola ) x 3 coexistence treatments, including intraclonal coexistence (+ G1-G1; +G2-G2) and interclonal coexistence (-G1-G2; + G1 + G2; +G1-G2 and -G1 + G2) and single clone (control treatment) (+ G1, -G1, +G2 and -G2). Homogeneity of variances and normality of data were examined using the Bartlett test and Shapiro-Wilk normality test respectively, and thus data were transformed when needed. Coexistence treatments (-G1-G2; + G1 + G2; +G1-G1; +G2-G2; +G1-G2 and -G1 + G2) were compared with Wilcoxon paired-matched non-parametric test, while for treatments + G1-G1 vs + G2-G2 and + G1-G2 vs -G1 + G2, the Mann-Whitney test was used. Differences in the mean absolute number of aphids in the different experimental treatments (Table 2 ) was studied as the change in the mean number of aphids over time using a generalized additive model (GAMs) with a Poisson distribution and smoothness estimation to fit models to data implemented in the R package “ mgcv ” (Wood 2018 ). Proportion of winged aphid among treatments were compared with proportion Z-test. All tests were performed using the R program (R Core Team 2018 ). Fresh weight of aphids was compared using a generalized linear model (GLM) with a Gaussian distribution incorporating as the genotype and endosymbiont factors and their interaction from the treatments with solitary colonies (+ G1; -G1; +G2 and -G2). The effect of the presence of endosymbionts and aphid genotype (fixed factors) were studied on the root/shoot ratio of plants with and without aphids (control treatment) using a generalized linear mixed model with a Gaussian distribution and as random factor the number total of aphids to fit models. Multiple comparisons of means for effects of models were made by Tukey Contrasts implemented in the R package “ multcomp ” (Hothorn et al. 2016 ). The different statistical models were compared using anova function and the effect of factors was determined using anova function implemented in the R package " car " (Fox et al. 2012 ). 3. RESULTS 3.2 Population growth rate (PGR) and population dynamic For the PGR variable, the GLM showed a significant effect for endosymbiont factor and the genotype x coexistence interaction (Table 3 ). As single colonies (control treatment), the clone G1 (infected and uninfected) showed a greater overall PGR than G2 (Fig. 1 ), whereas in the treatments of interclonal coexistence (+ G1 + G2, +G1-G2, -G1 + G2, and -G1-G2) the clone G2 outperformed the clone G1 (Fig. 1 ) regardless of endosymbiont-infection status. Intraclonal comparisons of coexisting colonies, either with or without harbouring R. insecticola not exhibited significant differences between clones (Fig. 1 ). A general effect of the endosymbiont R. insecticola on the mean PGR of aphids was observed, with a higher PGR in infected (0.052 ± 0.006 SE) than uninfected (0.025 ± 0.009 SE) aphids. Table 3 Generalized linear model performed for the variable PGR in aphid clones with different endosymbiont infection status and coexistence treatments. Source X 2 d.f. P value Genotype 0.600 1 0.438 Endosymbiont 7.038 1 0.007 ** Coexistence 0.289 2 0.865 Endosymbiont x Genotype 2.354 1 0.124 Endosymbiont x Coexistence 1.661 2 0.435 Genotype x Coexistence 69.955 2 6.448e-16 *** Endosymbiont x Genotype x Coexistence 5.614 3 0.060 The mean number of aphids significantly varied throughout the time globally (for all treatments) (F = 17.072, d.f. = 1.575; P value = 1.78e-06) and variation in the curve trends at treatment-level was observed (Table S1) (Fig. 2 ). Single separated colonies infected and uninfected as well intraclonal treatment of clone G1 (+ G1, -G1 and + G1-G1) showed an increased trend in the number of aphids from the half of the studied days (Fig. 2 ). On the contrary, the other treatments including intraclonal and interclonal coexistence and control treatments with the clone G2 showed a decreased trend in the aphid counts over the days since mid-period (Fig. 2 ). 3.2 Proportion of winged morphs and fresh weight of aphids The colonies of genotype G1, independently of the presence of R. insecticola , produced a lesser proportion of winged morphs (H = 60.06, P < 0.0001) than genotype G2 (Table 5 ). In the coexistence treatment + G1-G1, +G1 exhibited a greater proportion of winged morphs than -G1 (87.61 ± 7.69 ES and 12.14 ± 6.00 ES, respectively; W = 225.5, P < 0.001). Contrastingly, in treatment + G2-G2, -G2 exhibited a greater proportion of winged morphs than + G2 (63.76 ± 5.89 ES and 36.24 ± 5.89 ES, respectively; W = 67, P < 0.01). Finally, in coexistence treatments + G1 + G2 (9.27 ± 3.65 ES and 90.73 ± 3.65 ES, respectively; W = 30.84, P < 0.001), -G1-G2 (2.46 ± 1.69 ES and 97.54 ± 1.69 ES, respectively; W = 24.03, P < 0.001), +G1-G2 (7.87 ± 3.11 ES and 92.13 ± 3.11 ES, respectively; W = 28.99, P < 0.001) and -G1 + G2 (4.13 ± 3.05 ES and 95.87 ± 3.05 ES, respectively; W = 34.21, P < 0.001), clone G2 produced in all cases a greater number of winged morphs than G1, independently of the presence of R. insecticola (Table 5 ). Table 5 Total proportion of winged morphs and fresh weight (mean ± SE) of S. avenae aphids resulting from treatments and percentages of winged morphs corresponding to infected and uninfected with endosymbionts and aphid genotypes. Treatments N Fresh weight Proportion of winged * With R. insecticola (%) Without R. insecticola (%) G1 (%) G2 (%) +G1 20 0.53 ± 0.02 a 3.78 ± 0.50 ab -G1 20 0.21 ± 0.01 c 1.58 ± 0.28 a +G2 20 0.40 ± 0.01 b 21.91 ± 2.80 de -G2 20 0.45 ± 0.02 b 28.59 ± 2.45 e +G1-G1 20 - 9.96 ± 1.37 bc 87.61 ± 7.69** 12.14 ± 6.00 +G2-G2 20 - 24.03 ± 2.67 e 36.24 ± 5.89 63.76 ± 5.89** +G1 + G2 20 - 19.20 ± 2.63 de 9.27 ± 3.65 90.73 ± 3.65** +G1-G2 20 - 18.99 ± 2.45 de 7.87 ± 3.11 92.13 ± 3.11** -G1 + G2 20 - 21.63 ± 4.67 de 4.13 ± 3.05 95.87± 3.05** -G1-G2 20 - 16.87 ± 3.60 cd 2.46±1.69 97.54± 1.69** * Non-parametric test Kruskal-Wallis . The same letter on the bars indicates that the values are not significantly different ( Dunn's test , P < 0.05). ** Indicates that the values are significantly different Wilcoxon The weight of aphid genotypes was significantly affected by the endosymbiont presence showing a significant interaction between those factors (F = 104.689, df = 1, P value < 2.2e-16), as + G1 individuals exhibited a greater fresh weight than -G1 whereas + G2 and –G2 did not exhibited significant difference in their fresh weight (Table 5 ). 3.3 Relative change in root/shoot ratio Overall, plants attacked by aphids showed a significant higher mean of root/shoot ratio (1.22 ± 0.06 ES) than control treatment (without aphids) (0.77 ± 0.03 ES) ( \(\chi\) 2 = 12.654, df = 1, P value = 0.0003). On the other hand, a significative effect of the genotype of aphids was found ( \(\chi\) 2 = 7.293, df = 1, P value = 0.0007), where + G2 or –G2 exhibited a greater relative change in root/shoot ratio than clone + G1 or -G1 (Fig. 3 ). However, the endosymbiont-infection status ( \(\chi\) 2 = 1.660, df = 1, P value = 0.197) nor the genotype x endosymbiont interaction ( \(\chi\) 2 = 0.320, df = 1, P value = 0.571) did not show a significant effect in the GLMMs. 4. Discussion In our study, we evaluated the role of the facultative endosymbiont of aphids R. insecticola on intra and interclonal competitive interactions of aphids. Our experiments revealed that, regardless the presence of R. insecticola , in the cases of single colonies the clone G1 exhibited a higher PGR than clone G2. However, in competitive interclonal coexistence, G2 outperformed clone G1. These results suggest that aphid clones have differential features that allow more efficient performance as coexisting or solitary colonies, but not in both contexts. Surprisingly, clone G2 exhibited a higher PGR when coexisting with G1 than when developing alone or intraclonal coexistence. Variation in competitive abilities among aphid clones has been observed among clones for other aphid species such as the pea aphid, Acyrthosiphon pisum (Hazell et al. 2005 ), but not including the effect of facultative endosymbionts. Similar results were also observed between a mutant colour (yellow) and its original (green) aphid clone which regulated its reproductive rate depending on the relative density of self and non-self-clones, but in this study only one aphid genotype was studied (Li and Akimoto 2021 ), which involved only intraclonal competition. In this study, authors have suggested that aphid clones exposed to clonal coexistence and single rearing could alter their reproduction by discriminating between closely related and unrelated clones in mixed colonies. In Chile, S. avenae aphid shows strong signatures of obligated parthenogenesis and aphid clones are frequently observe over several seasons (Figueroa et al. 2005 ). Interestingly, the clone G1 and G2 of S. avenae used in our study were found to be common in wheat and oat plantations of centre-south of Chile (G1 and G4 respectively in Zepeda-Paulo et al. (2021). In the field, the clone G1 has been observed as the dominant clone at the beginning of the season compared to the other clones (including G2), but this decreased from the mid-season, on the contrary the clone G2 increased its prevalence throughout the season being dominant at the end of the season (Zepeda-Paulo et al. 2021). In light of our results, temporal dynamic and dominance of aphid clones throughout the growing season could be explained in part by a differential response of aphid clones, by example when they are exposed to lesser competitive (e.g., early season) and highly competitive scenarios (i.e., growing season), which would contribute to maintaining clonal variation within natural populations. The greater competitive ability of the clone G2 can be explained by a rapid occupation of the resource, developing a niche pre-emption strategy. This result supports Silvertown`s hypothesis (2004) for niche pre-emption limiting the diversification of late-arriving lineages. Evidence supporting this is the higher number of individuals of clone G2 than G1 at day 15 developing with or without harbouring R. insecticola , with clone G2 reaching an asymptote at a stage when clone G1 growing alone was initiating an exponential type of growth (Fig. 2 ). Consistent with our data, a similar study found that one clone of aphid species Myzus persicae outgrew another clone only under coexistence (Turcotte et al. 2011a , b ). Thus, aphid clones appear interact when mixed, leading to an asymmetrical competitive interaction, which in turn can drive eco-evolutionary feedback loops. However, in that work Turcotte et al. ( 2011a , b ) did not explore the mechanism underpinning clonal interaction. In our study, unlike our initial prediction, we can state that the asymmetric interaction between clone G1 and G2 appear not to be related with the presence of endosymbionts. Although competitive interactions between aphid clones was not significantly influenced by endosymbiont-infection, we have found that individual of S. avenae aphids harbouring the facultative endosymbiont R. insecticola exhibited a greater PGR than endosymbiont-free aphids. Furthermore, the positive effect on PGR that R. insecticola inflicts on the different aphid clones studied here agrees with the fact that positive selection of aphid clones harbouring R. insecticola in S. avenae natural populations (Zepeda-Paulo et al. 2021). Since high Regiella -infection levels were reached (87%-93%) over the course of a season on different cereal plants (wheat and oat) (Zepeda-Paulo et al. 2021), which shed some light on how facultative endosymbionts could drive different eco-evolutionary pathways of aphid clones at field level. For instance, the fact the clone G1 harbouring endosymbiont (+ G1) showed a higher fresh weight growing as solitary clone than -G1 and also than + G2 and -G2, suggest that R. insecticola positively affects the traits of this clone. This positive effect could be associated with an adaptation to host plants, similar to when the reproduction of infected pea aphids on clover is enhanced (Tsuchida et al. 2004 ), as a defensive effect of Regiella in the aphid S. avenae has already been ruled out (Zepeda-Paulo et al., 2017 ) Another interesting result is the effect of endosymbionts on the proportion of winged morphs in aphid clones exposed to intraclonal interactions. Herein we found that harbouring R. insecticola affects the proportion of winged individuals in a clone-dependent manner. While R. insecticola resulted in a lower final proportion of winged individuals in clone G1, this response changed when aphids were exposed to interclonal interactions, where clone G2 produced a higher proportion of winged morphs in response to clonal competence. Previous studies have confirmed that main factors inducing winged individuals in aphids are overcrowding and the presence of predation risk (Mehrparvar et al. 2013 ) and parasitism (Braendle et al. 2006 ). There is also genetic variation between clones in the winged morphs (Braendle et al., 2005 ). Mixed effects on the production of winged individuals due to the presence of R. insecticola have been described in the aphid pea, with evidence that support positive (Leonardo and Mondor 2006 ) and negative (Reyes et al. 2019 ), differences that have been attributed in part to the Regiella genotype (Reyes et al. 2019 ). A reduced production of winged individuals in aphids harbouring R. insecticola in a temperature-dependent manner was also described in S. avenea (Liu et al. 2019 ). Here we add that variation in winged production in aphids harbouring facultative endosymbionts also appears to depend on coexistence with other clones. We also inquired about the impact of the study clones, either with or without harbouring R. insecticola , on the relative change of root/shoot ratio in the host plant as a measured of plant stress induced by aphids. We observed that aphid infestation significantly increased the root/shoot ratio in wheat plants compared aphid-free plants. Feeding by aphids can affect plant growth and structure, mainly by reducing root tissue density, likely by a carbohydrate depletion in plants due to translocation from roots to shoots (Smith and Schowalter 2001 ). Interestingly, our results showed differences in the impact of aphids on the root/shoot ratio between aphid clones, indicating different levels of damage produced by the aphid clones. For instance, clone G1 showed a greater damaging capacity on host plants compared to clone G2, which could indicate for the aphid genotypes studied that clonal competition could be beneficial for plants as clone G2 outperformed clone G1 in competitive interaction. Although R. insecticola appears to do not induce significant changes on the root/shoot ratio, aphids harbouring R. insecticola showed a lesser damaging capacity on the host plants than endosymbiont-free aphids, which was true for both aphid clones studied. A similar tendency to show a lower root/shoot ratio in plants infected with aphids that harbour facultative endosymbionts was reported in the pea aphid- Hamiltonella defensa -broad bean system (Serteyn et al. 2020 ). Further studies are needed to understand how variation in the aphid genotype x endosymbiont interaction in different ecological contexts (e.g., intra and interclonal competition) affect growth, photosynthesis, and reproduction of host plants. . Conclusions The results obtained in this study showed that aphid clones varied considerably in their population growth rate when they are exposed to competitive and unrivalled environments. That trade-off in competitive interactions among aphid clones can influence the dynamics of aphid populations and impact on plant growth and structure. While facultative endosymbionts like R. insecticola do not play a significant role in directly mediating the competitive interactions of aphid clones or effecting specific plant traits, their presence does enhance aphid performance. Aphids harbouring R. insecticola showed higher growth rates in various coexistence scenarios and across different clones with a concurrently reduced capacity to damage host plants. These findings are particularly relevant in the context of pest management in cereal crops. Understanding the role of facultative endosymbionts in aphid dynamics offers new perspectives for developing more specific and sustainable pest control strategies. Leveraging the differential effects of R. insecticola on aphid competition and plant health could lead to more effective and ecologically conscious approaches for managing aphid populations in agricultural settings. Ultimately, our study underscores the importance of considering symbiotic relationships in pest management and contributes to a more nuanced understanding of the eco-evolutionary dynamics of pest species in agroecosystems. This ecological perspective is vital not only for predicting changes in pest populations but also for developing sustainable and environmentally friendly control methods. Declarations Author Contribution C.R. and M.M. Conceptualization; C.R. and M.M. Methodology; F.Z. and M.M. data collection; M.R. molecular analyses; M.M. and F.Z. formal data analysis; M.M. and M.R. writing - original draft preparation; C.R. and F.Z. writing—review and editing; C.R. and F.Z. funding. References Borodovsky M, McIninch J (1993) GENMARK: parallel gene recognition for both DNA strands. Computers and Chemistry 17(2):123-133 https://doi.org/10.1016/0097-8485(93)85004-V Braendle, C., Friebe, I., Caillaud, M. C., and Stern, D. L. (2005). Genetic variation for an aphid wing polyphenism is genetically linked to a naturally occurring wing polymorphism. Proceedings of the Royal Society B: Biological Sciences 272(1563): 657–664 https://doi.org/10.1098/rspb.2004.2995 Braendle C, Davis G K, Brisson J A, Stern DL (2006) Wing dimorphism in aphids. Heredity 97(3): 192-199 https://doi.org/10.1038/sj.hdy.6800863 Bruijning M, Jongejans E, Turcotte MM (2019) Demographic responses underlying eco‐evolutionary dynamics as revealed with inverse modelling. Journal of Animal Ecology 88(5):768-779 https://doi.org/10.1111/1365-2656.12966 Douglas AE (2009). The microbial dimension in insect nutritional ecology. Functional Ecology 23(1):38-47 https://doi.org/10.1111/j.1365-2435.2008.01442.x Ferrari J, Vavre F (2011) Bacterial symbionts in insects or the story of communities affecting communities. Philosophical Transactions of the Royal Society of London B: Biological Sciences 366(1569): 1389–1400 https://doi.org/10.1098/rstb.2010.0226 Feldhaar H (2011) Bacterial symbionts as mediators of ecologically important traits of insect hosts. Ecological Entomology 36:533-543 https://doi.org/10.1111/j.1365-2311.2011.01318.x Figueroa CC, Simon JG, Le Gallic JF, Prunier-Leterme N, Briones LM, Dedryver CA, Niemeyer HM (2005) Genetic structure and clonal diversity of an introduced pest in Chile, the cereal aphid Sitobion avenae. Heredity 95(1):24–33 https://doi.org/10.1038/sj.hdy.6800662 Fox J, Weisberg S, Adler D, Bates D, Baud-Bovy G, Ellison S, ... Heiberger R (2012) Package ‘car’. Vienna: R Foundation for Statistical Computing, 16. Hazell SP, Gwynn DM, Ceccarelli S, Fellowes MDE (2005) Competition and dispersal in the pea aphid: clonal variation and correlations across traits. Ecological Entomology 30(3):293-298 https://doi.org/10.1111/j.0307-6946.2005.00703.x Hertaeg C, Risse M, Vorburger C, De Moraes CM, Mescher MC (2021) Aphids harbouring different endosymbionts exhibit differences in cuticular hydrocarbon profiles that can be recognized by ant mutualists. Scientific Reports 11(1):19559. https://doi.org/10.1038/s41598-021-98098-2 Hothorn T, Bretz F, Westfall P, Heiberger RM, Schuetzenmeister A, Scheibe S, Hothorn M T (2016) Package ‘multcomp’. Simultaneous inference in general parametric models. Project for Statistical Computing, Vienna, Austria. Koga R, Tsuchida T, Fukatsu T (2003) Changing partners in an obligate symbiosis: a facultative endosymbiont can compensate for loss of the essential endosymbiont Buchnera in an aphid. Proceedings of the Royal Society B: Biological Sciences, 270(1533), 2543–2550. https://doi.org/10.1098/rspb.2003.2537 Koga R, Tsuchida T, Sakurai M, Fukatsu T (2007) Selective elimination of aphid endosymbionts: effects of antibiotic dose and host genotype, and fitness consequences. FEMS Microbiology Ecology, 60(2), 229–239. https://doi.org/10.1111/j.1574-6941.2007.00284.x Leigh SG, Emden HF van (2017) Population dynamics: cycles and patterns., CABI Books. CABI. https://doi.org/10.1079/9781780647098.0262. Leonardo TE, Mondor EB (2006) Symbiont modifies host life-history traits that affect gene flow. Proceedings of the Royal Society B: Biological Sciences 273(1590):1079-84. https://doi/10.1098/rspb.2005.3408 Li Y, Akimoto S I (2021) Self and non-self recognition affects clonal reproduction and competition in the pea aphid. Proceedings of the Royal Society B: Biological Sciences 288(1953): 20210787. https://doi.org/10.1098/rspb.2021.0787 Liu XD, Lei H X, Chen FF (2019) Infection pattern and negative effects of a facultative endosymbiont on its insect host are environment-dependent. Scientific Reports 9: 4013. https://doi/10.1038/s41598-019-40607-5 Llewellyn KS, Loxdale HD, Harrington R, Clark SJ, Sunnucks P (2004) Evidence for gene flow and local clonal selection in field populations of the grain aphid ( Sitobion avenae ) in Britain revealed using microsatellites. Heredity 93(2): 143-153. https://doi.org/10.1038/sj.hdy.6800466 Luo C, Chai R, Liu X, Dong Y, Desneux N, Feng Y, Hu Z (2022) The facultative symbiont Regiella insecticola modulates non-consumptive and consumptive effects of Harmonia axyridis on host aphids. Entomologia Generalis 42: 733-741. https://doi.org/10.1127/entomologia/2022/1368 Łukasik, P.; Dawid, M.A.; Ferrari, J.; Godfray, H.C.J. (2013) The diversity and fitness effects of infection with facultative endosymbionts in the grain aphid, Sitobion avenae . Oecologia 173: 985–996. McFall-Ngai M, Hadfield MG, Bosch T CG, Carey HV, Domazet-Lošo T, Douglas AE, et al. (2013) Animals in a bacterial world, a new imperative for the life sciences. Proceedings of the National Academy of Sciences of the United States of America 110(9): 3229-3236. https://doi.org/10.1073/pnas.1218525110 Mehrparvar M, Zytynska SE, Weisser W W (2013) Multiple cues for winged morph production in an aphid metacommunity. PLoS ONE 8(3): e58323. https://doi.org/10.1371/journal.pone.0058323 Moran NA (2006) Symbiosis. Current Biology 16(20): R866 - R871 Oliver KM, Russell JA, Moran NA, Hunter M S (2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proceedings of the National Academy of Sciences 100(4):1803-1807. https:// doi.org/10.1073/pnas.0335320100. Oliver K M, Degnan P H, Burke GR, Moran N A (2010) Facultative symbionts in aphids and the horizontal transfer of ecologically important traits. Annual Review of Entomology 55: 247–266. https://doi.org/10.1146/annurev-ento-112408-085305 Oliver K M, Campos J, Moran N A, Hunter M S (2008) Population dynamics of defensive symbionts in aphids. Proceedings of the Royal Society of London B: Biological Sciences 275(1632): 293–299. https://doi.org/10.1098/rspb.2007.1192 Oliver KM, 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(4): 1803–1807. https://doi.org/10.1073/pnas.0335320100 Peccoud J, Bonhomme J, Mahéo F, de la Huerta M, Cosson O, Simon JG (2014) Inheritance patterns of secondary symbionts during sexual reproduction of pea aphid biotypes. Insect Science 21(3): 291–300. https://doi.org/10.1111/1744-7917.12083 Petermann JS, Müller C B, Roscher C, Weigelt A, Weisser W W, Schmid B (2010) Plant species loss affects life-history traits of aphids and their parasitoids. PLoS ONE 5(8):e12053. https://doi.org/10.1371/journal.pone.0012053 Pons I, Renoz F, Hance T (2019) Fitness costs of the cultivable symbiont Serratia symbiotica and its phenotypic consequences to aphids in presence of environmental stressors. Evolutionary Ecology 33: 825-838. Pons I, Renoz F, Noël C, Hance T (2019b) Circulation of the cultivable symbiont Serratia symbiotica in aphids is mediated by plants. Frontiers in Microbiology 10: 764. https://doi.org/10.3389/fmicb.2019.00764 Kaech H, Jud S, Vorburger C (2022) Similar cost of Hamiltonella defensa in experimental and natural aphid‐endosymbiont associations. Ecology and Evolution 12(1): e8551. https://doi.org/10.1002/ece3.8551 R Core Team (2018) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. Ramírez-Cáceres G E, Moya-Hernández M G, Quilodrán M, Nespolo R F, Ceballos R, Villagra CA Ramírez, C C (2019) Harbouring the secondary endosymbiont Regiella insecticola increases predation risk and reproduction in the cereal aphid Sitobion avenae . Journal of Pest Science 92: 1039-1047. https://doi.org/10.1007/s10340-019-01090-z Reyes M L, Laughton A M, Parker B J, et al. (2019) The influence of symbiotic bacteria on reproductive strategies and wing polyphenism in pea aphids responding to stress. Journal of Animal Ecology 88: 601–611. https://doi.org.utalca.idm.oclc.org/10.1111/1365-2656.12942 Scarborough CL, Ferrari J, Godfray H C J (2005) Aphid protected from pathogen by endosymbiont. Science 310(5755): 1781. https://doi.org/10.1126/science.1120180 Schillewaert S, Parmentier T, Vantaux A, Van den Ende W, Vorburger C, Wenseleers T (2017) The influence of facultative endosymbionts on honeydew carbohydrate and amino acid composition of the black bean aphid Aphis fabae. Physiological Entomology 42(2): 125-133. https://doi.org/10.1111/phen.12181 Serteyn L, Quaghebeur C, Ongena M, Cabrera N, Barrera A, Molina-Montenegro MA, Francis F, Ramírez CC (2020) Induced systemic resistance by a plant growth-promoting rhizobacterium impacts development and feeding behavior of aphids. Insects 11(4):234. https://doi.org/10.3390/insects11040234 Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18(2): 233-234. https://doi.org/10.1038/72708 Simon J C, Baumann S, Sunnucks P, Hebert PD N, Pierre JS, Le Gallic JF, Dedryver C A (1999) Reproductive mode and population genetic structure of the cereal aphid Sitobion avenae studied using phenotypic and microsatellite markers. Molecular Ecology 8(4): 531-545. https://doi.org/10.1046/j.1365-294x.1999.00583.x Simon JG, Boutin S, Tsuchida T, Koga R, Le Gallic JF, Frantz A, Fukatsu T (2011) Facultative symbiont infections affect aphid reproduction. PLoS ONE 6(7): e21831. https://doi.org/10.1371/journal.pone.0021831 Smith J P, Schowalter TD (2001) Aphid‐induced reduction of shoot and root growth in Douglas‐fir seedlings. Ecological Entomology 26(4): 411-416. Sudakaran S, Kost C, Kaltenpoth M (2017) Symbiont acquisition and replacement as a source of ecological innovation. Trends in Microbiology, 25(5), 375-390. https://doi:org/10.1016/j.tim.2017.02.014. Sunnucks P, Hales D F (1996) Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae). Molecular Biology and Evolution 13(3): 510-524. https://doi.org/10.1093/oxfordjournals.molbev.a025612 Thomas S, Dogimont C, Boissot N (2011) Association between Aphis gossypii genotype and phenotype on melon accessions. Arthropod-Plant Interactions 6(1): 93–101. https://doi.org/10.1007/s11829-011-9155-2 Tsuchida T, Koga R, Fukatsu T (2004) Host plant specialization governed by facultative symbiont. Science 303(5666): 1989. https://doi.org/10.1126/science.1094611 Tsuchida T, Koga R, Horikawa M, Tsunoda T, Maoka T, Matsumoto S, Simon JG, Fukatsu T (2010) Symbiotic bacterium modifies aphid body color. Science 330(6007): 1102-1104. https://doi.org/10.1126/science.1195463 Turcotte M M, Reznick DN, Hare J D (2011a) Experimental assessment of the impact of rapid evolution on population dynamics. Evolutionary Ecology Research 13(2): 113–131. https://doi.org/10.1111/j.1461-0248.2011.01676.x Turcotte MM, Reznick D N, Hare J D (2011b) The impact of rapid evolution on population dynamics in the wild: experimental test of eco-evolutionary dynamics. Ecology Letters 14(11): 1084–1092. https://doi.org/10.1111/j.1461-0248.2011.01676.x Vorburger C, Perlman S J (2018) The role of defensive symbionts in host–parasite coevolution. Biological Review 93:1747-1764. https://doi.org/10.1111/brv.12417 Wagner SM, Martinez A J, Ruan YM, Kim K L, Lenhart P A, Dehnel A C, Oliver KM, White J A (2015) Facultative endosymbionts mediate dietary breadth in a polyphagous herbivore. Functional Ecology 29(11): 1402–1410. https://doi.org/10.1111/1365-2435.12459 Wilson A C C, Massonnet B, Simon JG, Prunier-Leterme N, Dolatti L, Llewellyn K S, Figueroa C C, Ramírez C C, Blackman R L, Estoup A, Sunnucks P (2004) Cross-species amplification of microsatellite loci in aphids: assessment and application. Molecular Ecology Notes 4(1): 104–109. https://doi.org/10.1046/j.1471-8286.2004.00584.x Wood S (2018) Mixed GAM computation vehicle with automatic smoothness estimation. R package version 1.8-24. Xin J-J, Shang Q-L, Desneux N, Gao X-W (2014) Genetic diversity of Sitobion avenae (Homoptera: Aphididae) populations from different geographic regions in China. PLoS ONE 9(10): e109349. https://doi.org/10.1371/journal.pone.0109349 Zepeda-Paulo F, Lavandero B (2021) Effect of the genotypic variation of an aphid host on the endosymbiont associations in natural host populations. Insects 12(3): 217. https://doi.org/10.3390/insects12030217 Zepeda‐Paulo FA, Villegas C, Lavandero B (2017) Host genotype–endosymbiont associations and their relationship with aphid parasitism at the field level. Ecological Entomology 42(1): 86-95. https://doi.org/10.1111/een.12361 Additional Declarations No competing interests reported. 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Ramírez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDACdhBhAGI0AIkCEI+5Ab8WZpgWngOMDWAGAyMxWkBAIoFILfzNvA8/3Si4Jyc/8435gx8GdvIM7Afxa5E4zG4snWNQbMw4O8ewsccg2bCBJxG/FgNmNgagloTEZukcoGoDoIckCDgMqIX5N1BLfZvkGcPGPwYH7InRwgayJYFHgsewGWhLIkEtEofZ2KyBWgxn8KQVzpYxSE5uI+QX/vY25ts5fxLk5dsPb/j4psLOtp/98AG8WjABG4nqR8EoGAWjYBRgAQC4sTsvNzMqQAAAAABJRU5ErkJggg==","orcid":"","institution":"Universidad de Talca","correspondingAuthor":true,"prefix":"","firstName":"Claudio","middleName":"C.","lastName":"Ramírez","suffix":""}],"badges":[],"createdAt":"2024-03-06 13:47:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4021194/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4021194/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52422827,"identity":"bcfc279d-5019-49ba-8b83-5f7a8937cb10","added_by":"auto","created_at":"2024-03-11 13:02:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":223744,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation growth rate (PGR) of two genotypes of the aphid \u003cem\u003eS. avenae\u003c/em\u003e (G1 and G2), reared as single clones harbouring (+) or not-harbouring (-) the facultative endosymbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e in the different coexistence treatments (intra-clonal, inter-clonal and control).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4021194/v1/581142d2e2fb3e4675c0c03f.png"},{"id":52422826,"identity":"ea6ff283-2d14-4c48-8fdc-298409b3c0fe","added_by":"auto","created_at":"2024-03-11 13:02:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":518748,"visible":true,"origin":"","legend":"\u003cp\u003eAphid growth curves of two genotypes of the aphid \u003cem\u003eS. avenae\u003c/em\u003e (G1 and G2), reared as single clones harbouring (+) or not-harbouring (-) the facultative endosymbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e and different coexistence treatments (intraclonal and interclonal).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4021194/v1/556b3ead2dff2838845b51a8.png"},{"id":52422825,"identity":"e3d172b9-5f8f-4186-9e68-ee4fd61e3e29","added_by":"auto","created_at":"2024-03-11 13:02:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198853,"visible":true,"origin":"","legend":"\u003cp\u003eRelative change in root/shoot ratio (mean ± SE) of wheat seedling of the two genotypes of the aphid \u003cem\u003eS. avenae\u003c/em\u003e (G1 and G2) harbouring (+) or not-harbouring (-) the facultative endosymbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4021194/v1/3c8eb7bd5c4d38188e25193b.png"},{"id":57350615,"identity":"3d03e07c-95f9-4efa-ae8c-b521a11bf492","added_by":"auto","created_at":"2024-05-29 12:48:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1710377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4021194/v1/095fdda2-858e-4669-aba1-1edd960b13d4.pdf"},{"id":52422824,"identity":"e91e637b-d301-440f-916b-9f4bb87b779d","added_by":"auto","created_at":"2024-03-11 13:02:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47169,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4021194/v1/ecc6e596de429c919f587c42.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Are competitive interactions between aphid clones mediated by facultative endosymbionts?","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eAnimal symbionts can play a decisive role in determining the outcome of ecological and evolutionary processes (Moran \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; McFall-Ngaia et al. 2013; Vorburger and Perlman \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, symbiosis has allowed many animal species to acquire novel functions, occupy new ecological niches, and influence their ecological interactions with other organisms (Oliver et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Scarborough et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sudakaran et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While the impact of symbiosis has been mostly studied in mutualistic and predatory ecological interactions (Douglas \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Feldhaar \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), little is known about its role in competitive interactions.\u003c/p\u003e \u003cp\u003eAphids are phytophagous insects with worldwide distribution, representing an important agricultural pest of several crops (Leight and Emden 2017). Bacterial endosymbionts have been shown to play a key role in aphid biology (Oliver et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Aphids are hosts of a primary endosymbiont, \u003cem\u003eBuchnera aphidicola\u003c/em\u003e, a bacterium that is vertically transmitted and synthesizes essential amino acids and other nutrients, which are necessary for aphid reproduction and survival (Koga et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Moreover, common associations between aphids and several secondary or facultative bacterial endosymbionts have been evidenced in different aphid populations. Facultative endosymbionts are predominantly vertically transmitted from females to offspring but also horizontal transfer occasionally could occur within and between species (Ferrari and Vavre \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Oliver et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Harbouring endosymbionts may directly influence host biology by conferring conditional benefits and/or fitness costs on hosts(Ferrari and Vavre \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pons et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003ea; Kaech et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the most well-known endosymbiont-mediated effects on aphids is the protection against parasitoids, such when the pea aphid (\u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e) is infected with the facultative endosymbiont \u003cem\u003eHamiltonella defense\u003c/em\u003e (Oliver et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Moreover, the facultative endosymbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e protects to the pea aphid \u003cem\u003eA. pisum\u003c/em\u003e from the lethal fungus \u003cem\u003ePandora neoaphidis\u003c/em\u003e (Scarborough et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). According to Tsuchida et al., (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), pea aphids harbour \u003cem\u003eR. insecticola\u003c/em\u003e improves significantly its performance on clover (\u003cem\u003eTrifolium\u003c/em\u003e sp.). Besides, facultative endosymbionts can indirectly shape ecological interactions by altering traits that influence the mutualistic and antagonist relationships of aphids. For instance, endosymbionts can change the honeydew and cuticular composition of aphids, which can affect interspecific communication with their mutualistic ants (Schillewaert et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hertaeg et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, the predatory activity of ladybirds on aphids can be altered by harboring facultative endosymbionts, either positively or negatively (Ram\u0026iacute;rez-C\u0026aacute;ceres et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, facultative endosymbiont can influence the trophic relationships such as aphid-host plant interactions. In this sense, Wagner et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) showed that despite the low clonal variability in the aphid \u003cem\u003eAphis craccivora\u003c/em\u003e, the presence or absence of maternal inhered facultative endosymbionts can act as facilitators for the expansion or restriction of aphid clones on new plant hosts. In addition, endosymbionts can be horizontally transferred between aphids and host plants, entering plants, and modify the functioning of the plant by promote the root growth (larger Root: shoot) (Pons et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Thereby, aphid clone-facultative endosymbiont associations could be modulating the relative success and the ecological interactions of aphid clones on their host plants.\u003c/p\u003e \u003cp\u003eAlthough facultative endosymbiont-mediated effects on aphids have been widely studied in several aphid species, little is known about the role of facultative endosymbionts on the competitive interactions among aphid clones. Due to its predominant asexual reproduction (parthenogenetic), aphid populations are commonly conformed by a clonal genotype set subjected to diverse ecological stressors and seasonal changes in their environment. Furthermore, variability and rapid seasonal shifts in the frequency of endosymbiont infection have been reported among aphid clones within-populations (Zepeda-Paulo et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, 2021). Interclonal interactions could play an important role in the understating of mechanisms that facilitate the coexistence of clones within aphid pest species (Bruijninget al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), however the effect of facultative endosymbionts on the competitive interactions of aphids has been little aimed. For example, Turcotte et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e) evaluated the population dynamics of three clones of the green peach aphid (\u003cem\u003eM. persicae\u003c/em\u003e s. \u003cem\u003estr.\u003c/em\u003e), observing that two of the three clones studied increased their population growth rate between 28\u0026ndash;34% under interclonal competition compared to the control treatment (without interclonal competition). A similar study carried out with those clones in the field showed an increase in the population growth rate of 47% and a 67% increase in the population under interclonal competition (Turcotte et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e). These studies suggest that interclonal interactions could be common within aphid populations, however to our knowledge there is no published evidence on how facultative endosymbionts affect the inter and intra-clonal interactions in the aphid group, such as when different clones coexist in the same host plant.\u003c/p\u003e \u003cp\u003eIn Chile, the populations of \u003cem\u003eSitobion avenae\u003c/em\u003e exhibit a low genetic variability, characterized by a few predominant clones that are able to successfully colonize different cultivated and wild host plants through different climatic zones, which have been maintained over time (\u0026ldquo;superclones\u0026rdquo;) (Figueroa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The success of these abundant and persistent clones in the field is likely affected by specific associations of aphid clones and endosymbionts found in the field (Zepeda-Paulo et al. 2021). Also, in this aphid species has been discarded a protection-mediated role by facultative endosymbionts (\u003cem\u003eH. defensa\u003c/em\u003e and \u003cem\u003eR. insecticola\u003c/em\u003e) against parasitoids (Łukasik et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zepeda-Paulo et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given that the presence of facultative endosymbionts usually generates positive effects on the individuals that host them, our working hypothesis was that the coexistence of \u003cem\u003eS. avenae\u003c/em\u003e clones is modulated by the presence of the facultative endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e. Thus, the aim of this study was to analyse the biological effects of facultative endosymbionts on the ecological interactions of aphid clones to answer the following question: what is the impact of facultative endosymbiosis on the intra and interclonal competitive interactions of aphid clones? and to what extent does the outcome of competitive interactions depend on the genotypic composition of the interacting clones? These questions were addressed by subjecting the two prevalent clones of the aphid \u003cem\u003eS. avenae\u003c/em\u003e, both infected and uninfected with the most common facultative endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e, rearing under competition on a common host plant and evaluates aphid population growth rates and life-history traits, including the consequences on functional traits of the host plants (root/shoot ratio).\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Aphid sampling and rearing conditions\u003c/h2\u003e \u003cp\u003eTwo genotypes of the aphid \u003cem\u003eS. avenae\u003c/em\u003e were established from field as clonal lineages and assayed in the present study. Multilocus genotypes were determined by amplifying eight microsatellite loci described in \u003cem\u003eS. avenae\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The two aphid genotypes (clone G1 and G2) hereafter used in this study corresponded to individuals collected on wheat fields with a widespread distribution and frequency across different agroclimatic zones sampled (Zepeda-Paulo et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEndosymbiont status and multilocus genotype of clones of the cereal aphid \u003cem\u003eSitobion avenae\u003c/em\u003e based on eight microsatellite loci used in the coexistence experiments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClone\u003c/p\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndosymbiont\u003c/p\u003e \u003cp\u003estatus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSm11\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS3.43\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS16b\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eS30\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eS4Σ\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eS5L\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eSm17\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eSm10\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRegiella insecticola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e224226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e177179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e175181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e243245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e115115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e185187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e-G1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDisinfected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e224226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e177179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e175181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e243245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e115115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e185187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRegiella insecticola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e205205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e177179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e181185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e241245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e115115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e185185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e-G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaturally uninfected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e160164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e205205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e177179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e181185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e241245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e115115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e185185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlive specimens were transferred to the laboratory and composed separately clonal lineages. The colony of clone G1 was collected naturally infected with \u003cem\u003eR. insecticola\u003c/em\u003e, was split in two parts, one part was disinfected with antibiotics following the procedure described by Koga et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Simon et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and the other part was kept as collected. This allowed the rearing of G1 with and without the presence of \u003cem\u003eR. insecticola\u003c/em\u003e (hereafter\u0026thinsp;+\u0026thinsp;G1 and \u0026ndash;G1, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Differently, the aphid genotype G2 was collected both clones naturally harbouring and uninfected the \u003cem\u003eR. insecticola\u003c/em\u003e (hereafter\u0026thinsp;+\u0026thinsp;G2 and \u0026ndash;G2, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All aphid clones, infected and uninfected with \u003cem\u003eR. insecticola\u003c/em\u003e, were independently maintained in the laboratory on potted plants of wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e; cv. Pantera-INIA) in rearing cages (50 x 40 x 30 cm) under controlled climatic conditions at a temperature of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026deg; C and 16L: 8D photoperiod and watered every three days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Genotyping and endosymbiont detected in aphid samples\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from each aphid using the \u0026ldquo;salting-out\u0026rdquo; method (Sunnucks and Hales \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Each aphid was genotyped with eight discriminant microsatellite loci (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These microsatellites have been previously isolated from \u003cem\u003eS. avenae\u003c/em\u003e by Wilson et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and extensively used to determine the genetic diversity worldwide (Simon et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Llewellyn et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Figueroa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Xin et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The microsatellite loci were amplified modifying the forward primer with M13(-21) tail at the 5\u0026rsquo; end for each microsatellite. The reaction was performed adding the sequence-specific forward primer with M13(-21) tail at its 5\u0026prime; end, the sequence-specific reverse primer and the universal FAM or VIC or NED fluorescent-labeled M13(-21) primer (Schuelke, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The PCR products were genotyped in Macrogen Inc (Rep. of Korea) and the electropherograms obtained were analysed using the in GENMARK version 1.3 (Borodovsky and McIninch \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In other hand, the presence of secondary endosymbionts was determined by molecular detection in each aphid. Using a PCR multiplex (Peccoud et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) for eight facultative endosymbionts in each clone, for all treatment. In this sense, first standardizing simplex methods PCR for all loci, using the obligatory bacterial endosymbiont of aphids, \u003cem\u003eBuchnera aphidicola\u003c/em\u003e, as positive control for PCR reaction. Also, in multiplex PCR were used samples of the aphid \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e (with known endosymbiont infections) as positive control in each amplification diagnostic. Polymerase chain reaction (PCR) amplifications were carried out in an Viriti Thermal Cycler (Applied Biosystems), under the following conditions: 5 min of denaturation at 94\u0026deg;C followed by 30 cycles with 30 s of denaturation at 94\u0026deg;C, 30 s at 58\u0026deg;C of annealing temperature and 1 min of elongation at 72\u0026deg;C and a ended with 5 min elongation at 72\u0026deg;C. Sequences were visualized through electrophoresis agarose gel (1.5%), using a GeneRuler 100 bp Plus DNA Ladder (Fermentas) for determining the size of each sequence. Each facultative endosymbiont was identified according to the size of the sequence in base pairs (bp) obtained from the total DNA of each individual aphid (Peccoud et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The following diagnostic sizes were used: \u003cem\u003eSpiroplasma\u003c/em\u003e (1500 bp), \u003cem\u003eSerratia symbiotica\u003c/em\u003e (1140 bp), \u003cem\u003eRegiella insecticola\u003c/em\u003e (840 bp), \u003cem\u003eRickettsia\u003c/em\u003e (600 bp), \u003cem\u003eHamiltonella defensa\u003c/em\u003e (480 bp), \u003cem\u003eFukatsuia symbiotica\u003c/em\u003e (500 bp) and \u003cem\u003eRickettsiella\u003c/em\u003e sp (300 bp) and \u003cem\u003eBuchnera aphidicola\u003c/em\u003e (270 bp) (Peccoud et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). All detections of endosymbionts were positive for \u003cem\u003eBuchnera aphidicola\u003c/em\u003e, in this sense the quality of DNA was enough. For the case of the detection in clones\u0026thinsp;+\u0026thinsp;G1 and +\u0026thinsp;G2 we only detected \u003cem\u003eR. insecticola\u003c/em\u003e and no other secondary endosymbionts was reported for our colonies of \u003cem\u003eS. avenae\u003c/em\u003e for all treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experiments design\u003c/h2\u003e \u003cp\u003eTo study the influence of facultative endosymbionts on the reproduction and competition of aphid clones, we performed a 2 x 2 x 2 experimental design to examinate the population growth of two aphid genotypes (G1 and G2) with [+] and without [-] the presence of the endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e exposed to two coexistence treatments; i) single aphid clone (+\u0026thinsp;G1, -G1, +G2 and -G2), and ii) a clonal mixing (G1-G1, +G2-G2,\u0026thinsp;+\u0026thinsp;G1\u0026thinsp;+\u0026thinsp;G2, +G1-G2, -G1\u0026thinsp;+\u0026thinsp;G2 and -G1-G2). The factors studied were genotype (G1 and G2), endosymbiont (with [+] and without [-] the presence of \u003cem\u003eR. insecticola\u003c/em\u003e) and coexistence status (single and coexisting genotypes). The factors were combined originating ten treatments with twenty replicates either one (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Each replicate was composed of a microcosm, which included three wheat seedlings grow in a pot (650 cc) containing a substrate composed by 1:1 (vermiculite: perlite). Each pot was protected using a transparent breezy plastic tube with nylon mesh-covered openings. All treatments the aphid colonies started with age-synchronized aphids. For this, 50 individuals of one genotype of approximately the same age were taken from the rearing cages and placed on wheat. After 24 hours, all adult aphids were removed, and only new-born nymphs were kept. The individuals starting the colonies in all the treatments were taken from this stock of age-synchronized nymphs. In treatments composed by solitary colonies (+\u0026thinsp;G1, -G1, +G2 and -G2), four nymphs were placed on the wheat seedlings in each microcosm. In treatments of coexisting aphids, two nymphs per genotype /infection status were used (G1-G1, +G2-G2,\u0026thinsp;+\u0026thinsp;G1\u0026thinsp;+\u0026thinsp;G2, +G1-G2, -G1\u0026thinsp;+\u0026thinsp;G2 and -G1-G2). This allowed the use of a similar density of aphids per microcosm in all ten treatments. Aphid numbers were counted by visual inspection at 0, 7, 15 and 26 days in the different treatments, similarly to reported by Turcotte et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e). At day 26, all aphids were removed and place on a white tray with alcohol 95% for a detailed counting, including all nymphs, winged and wingless adults. In the case of coexisting colonies, because aphids of different genotypes or harbouring \u003cem\u003eR. insecticola\u003c/em\u003e were not able to be distinguished phenotypically, individuals were genotyped by microsatellite loci and endosymbiont PCR-detected. Due to the large number of specimens recovered at the end of the experiment, the microsatellite genotyping and PCR-based endosymbiont detection was performed to a sub-sampled containing the 50% of the adults (wingless and winged aphids) obtained from each pot at the end experiment (day 26).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental design: the coexistence treatments are termed according to the presence of the aphid genotypes (G1 and G2) with (+) and without (-) the presence of the facultative endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCoexistence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of replicates\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e-G1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e-G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G1-G1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntraclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G2-G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntraclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G1\u0026thinsp;+\u0026thinsp;G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInterclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e+G1-G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInterclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e-G1\u0026thinsp;+\u0026thinsp;G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInterclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e-G1-G2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInterclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Population growth rate (PGR) and population dynamic\u003c/h2\u003e \u003cp\u003eThe population growth rate (PGR) in the different treatments was estimated as ln (Nf\u0026thinsp;+\u0026thinsp;0.001) - ln (Ni) / (tf \u0026ndash; ti), where Nf and Ni refers the final and initial number of aphids, respectively, and (tf \u0026ndash; ti) is the number of days of the experiment (26 days) (Thomas et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The 0.001 was added to Nf because in some replicates final numbers were zero, as recommended by Thomas et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The initial population size was 4 individuals in all replicates. In addition, the total number of aphids was studied at days 0, 7, 15 and 26 in the different treatments allowing the assessment of the temporal variation in the abundance of aphids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fresh weight and proportion of winged aphids\u003c/h2\u003e \u003cp\u003eTo evaluate whether the facultative endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e influenced aphid body mass, as described in other aphid systems (Oliver et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), 30 adult individuals of each genotype harbouring or not harbouring \u003cem\u003eR. insecticola\u003c/em\u003e were carefully identified and selected using a Zeiss Stemi DV4stereomicroscope, and then the weight was measured using a Mettler Toledo XS3DU microbalance. The proportion of winged aphid was calculated by dividing the density of winged individuals by the total aphid density (Petermann et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Relative root/shoot of plants\u003c/h2\u003e \u003cp\u003eThe root/shoot ratio of wheat seedling used for feeding aphids in the 10 treatments was calculated as the ratio of total root dry mass to total leaf dry mass. Once the experiment finished at day 26, each plant was dried at 60\u0026deg;C for 5 days and weighed and dry weight was recorded. An additional treatment composed by 20 control plants was also implemented. The relative change in the plant root/shoot ratio of each treatment was obtained by calculating the coefficient between the root/shoot ratio of each treatment and control plants using a microbalance Radwag.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Data analysis\u003c/h2\u003e \u003cp\u003ePGR data were transformed to antilog (exp) and compared using a generalized linear model (GLM) with a Gaussian distribution. PGR data analysis was performed on the experimental design contemplating 2 (genotypes G1 and G2) x 2 (with and without \u003cem\u003eR. insecticola\u003c/em\u003e) x 3 coexistence treatments, including intraclonal coexistence (+\u0026thinsp;G1-G1; +G2-G2) and interclonal coexistence (-G1-G2;\u0026thinsp;+\u0026thinsp;G1\u0026thinsp;+\u0026thinsp;G2; +G1-G2 and -G1\u0026thinsp;+\u0026thinsp;G2) and single clone (control treatment) (+\u0026thinsp;G1, -G1, +G2 and -G2). Homogeneity of variances and normality of data were examined using the Bartlett test and Shapiro-Wilk normality test respectively, and thus data were transformed when needed. Coexistence treatments (-G1-G2;\u0026thinsp;+\u0026thinsp;G1\u0026thinsp;+\u0026thinsp;G2; +G1-G1; +G2-G2; +G1-G2 and -G1\u0026thinsp;+\u0026thinsp;G2) were compared with Wilcoxon paired-matched non-parametric test, while for treatments\u0026thinsp;+\u0026thinsp;G1-G1 vs\u0026thinsp;+\u0026thinsp;G2-G2 and +\u0026thinsp;G1-G2 vs -G1\u0026thinsp;+\u0026thinsp;G2, the Mann-Whitney test was used. Differences in the mean absolute number of aphids in the different experimental treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was studied as the change in the mean number of aphids over time using a generalized additive model (GAMs) with a Poisson distribution and smoothness estimation to fit models to data implemented in the R package \u0026ldquo;\u003cem\u003emgcv\u003c/em\u003e\u0026rdquo; (Wood \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Proportion of winged aphid among treatments were compared with proportion Z-test. All tests were performed using the R program (R Core Team \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Fresh weight of aphids was compared using a generalized linear model (GLM) with a Gaussian distribution incorporating as the genotype and endosymbiont factors and their interaction from the treatments with solitary colonies (+\u0026thinsp;G1; -G1; +G2 and -G2). The effect of the presence of endosymbionts and aphid genotype (fixed factors) were studied on the root/shoot ratio of plants with and without aphids (control treatment) using a generalized linear mixed model with a Gaussian distribution and as random factor the number total of aphids to fit models. Multiple comparisons of means for effects of models were made by Tukey Contrasts implemented in the R package \u0026ldquo;\u003cem\u003emultcomp\u003c/em\u003e\u0026rdquo; (Hothorn et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The different statistical models were compared using anova function and the effect of factors was determined using anova function implemented in the R package \"\u003cem\u003ecar\u003c/em\u003e\" (Fox et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.2 Population growth rate (PGR) and population dynamic\u003c/h2\u003e\n \u003cp\u003eFor the PGR variable, the GLM showed a significant effect for endosymbiont factor and the genotype x coexistence interaction (Table\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). As single colonies (control treatment), the clone G1 (infected and uninfected) showed a greater overall PGR than G2 (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e), whereas in the treatments of interclonal coexistence (+\u0026thinsp;G1\u0026thinsp;+\u0026thinsp;G2, +G1-G2, -G1\u0026thinsp;+\u0026thinsp;G2, and -G1-G2) the clone G2 outperformed the clone G1 (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e) regardless of endosymbiont-infection status. Intraclonal comparisons of coexisting colonies, either with or without harbouring \u003cem\u003eR. insecticola\u003c/em\u003e not exhibited significant differences between clones (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). A general effect of the endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e on the mean PGR of aphids was observed, with a higher PGR in infected (0.052\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 SE) than uninfected (0.025\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 SE) aphids.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eGeneralized linear model performed for the variable PGR in aphid clones with different endosymbiont infection status and coexistence treatments.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ed.f.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndosymbiont\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoexistence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.865\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndosymbiont x Genotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.354\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.124\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndosymbiont x Coexistence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.435\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGenotype x Coexistence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69.955\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.448e-16 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndosymbiont x Genotype x Coexistence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe mean number of aphids significantly varied throughout the time globally (for all treatments) (F\u0026thinsp;=\u0026thinsp;17.072, d.f. = 1.575; \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;1.78e-06) and variation in the curve trends at treatment-level was observed (Table S1) (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e). Single separated colonies infected and uninfected as well intraclonal treatment of clone G1 (+\u0026thinsp;G1, -G1 and +\u0026thinsp;G1-G1) showed an increased trend in the number of aphids from the half of the studied days (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e). On the contrary, the other treatments including intraclonal and interclonal coexistence and control treatments with the clone G2 showed a decreased trend in the aphid counts over the days since mid-period (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.2 Proportion of winged morphs and fresh weight of aphids\u003c/h2\u003e\n \u003cp\u003eThe colonies of genotype G1, independently of the presence of \u003cem\u003eR. insecticola\u003c/em\u003e, produced a lesser proportion of winged morphs (H\u0026thinsp;=\u0026thinsp;60.06, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than genotype G2 (Table\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e). In the coexistence treatment\u0026thinsp;+\u0026thinsp;G1-G1, +G1 exhibited a greater proportion of winged morphs than -G1 (87.61 \u0026plusmn; 7.69 ES and 12.14 \u0026plusmn; 6.00 ES, respectively; W\u0026thinsp;=\u0026thinsp;225.5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Contrastingly, in treatment\u0026thinsp;+\u0026thinsp;G2-G2, -G2 exhibited a greater proportion of winged morphs than +\u0026thinsp;G2 (63.76 \u0026plusmn; 5.89 ES and 36.24 \u0026plusmn; 5.89 ES, respectively; W\u0026thinsp;=\u0026thinsp;67, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Finally, in coexistence treatments\u0026thinsp;+\u0026thinsp;G1\u0026thinsp;+\u0026thinsp;G2 (9.27 \u0026plusmn; 3.65 ES and 90.73 \u0026plusmn; 3.65 ES, respectively; W\u0026thinsp;=\u0026thinsp;30.84, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), -G1-G2 (2.46 \u0026plusmn; 1.69 ES and 97.54 \u0026plusmn; 1.69 ES, respectively; W\u0026thinsp;=\u0026thinsp;24.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), +G1-G2 (7.87 \u0026plusmn; 3.11 ES and 92.13 \u0026plusmn; 3.11 ES, respectively; W\u0026thinsp;=\u0026thinsp;28.99, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and -G1\u0026thinsp;+\u0026thinsp;G2 (4.13 \u0026plusmn; 3.05 ES and 95.87 \u0026plusmn; 3.05 ES, respectively; W\u0026thinsp;=\u0026thinsp;34.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), clone G2 produced in all cases a greater number of winged morphs than G1, independently of the presence of \u003cem\u003eR. insecticola\u003c/em\u003e (Table\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eTotal proportion of winged morphs and fresh weight (mean \u0026plusmn; SE) of \u003cem\u003eS. avenae\u003c/em\u003e aphids resulting from treatments and percentages of winged morphs corresponding to infected and uninfected with endosymbionts and aphid genotypes.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFresh weight\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProportion of winged *\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWith\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eR. insecticola\u003c/em\u003e (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWithout\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eR. insecticola\u003c/em\u003e (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG1 (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eG2 (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+G1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.53 \u0026plusmn; 0.02 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.78 \u0026plusmn; 0.50 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-G1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21 \u0026plusmn; 0.01 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.58 \u0026plusmn; 0.28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.40 \u0026plusmn; 0.01 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.91 \u0026plusmn; 2.80 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45 \u0026plusmn; 0.02 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.59 \u0026plusmn; 2.45 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+G1-G1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.96 \u0026plusmn; 1.37 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.61 \u0026plusmn; 7.69**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.14 \u0026plusmn; 6.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+G2-G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.03 \u0026plusmn; 2.67 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.24 \u0026plusmn; 5.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.76 \u0026plusmn; 5.89**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+G1\u0026thinsp;+\u0026thinsp;G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.20 \u0026plusmn; 2.63 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.27 \u0026plusmn; 3.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.73 \u0026plusmn; 3.65**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+G1-G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.99 \u0026plusmn; 2.45 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.87 \u0026plusmn; 3.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.13 \u0026plusmn; 3.11**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-G1\u0026thinsp;+\u0026thinsp;G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.63 \u0026plusmn; 4.67 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.13 \u0026plusmn; 3.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.87\u0026plusmn; 3.05**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-G1-G2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.87 \u0026plusmn; 3.60 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.46\u0026plusmn;1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.54\u0026plusmn; 1.69**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e* Non-parametric test \u003cem\u003eKruskal-Wallis\u003c/em\u003e. The same letter on the bars indicates that the values are not significantly different (\u003cem\u003eDunn\u0026apos;s test\u003c/em\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e** Indicates that the values are significantly different \u003cem\u003eWilcoxon\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe weight of aphid genotypes was significantly affected by the endosymbiont presence showing a significant interaction between those factors (F\u0026thinsp;=\u0026thinsp;104.689, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16), as +\u0026thinsp;G1 individuals exhibited a greater fresh weight than -G1 whereas +\u0026thinsp;G2 and \u0026ndash;G2 did not exhibited significant difference in their fresh weight (Table\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.3 Relative change in root/shoot ratio\u003c/h2\u003e\n \u003cp\u003eOverall, plants attacked by aphids showed a significant higher mean of root/shoot ratio (1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ES) than control treatment (without aphids) (0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ES) (\u003cspan\u003e\u003cspan\u003e\\(\\chi\\)\u003c/span\u003e\u003c/span\u003e\u003csup\u003e2\u003c/sup\u003e = 12.654, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.0003). On the other hand, a significative effect of the genotype of aphids was found (\u003cspan\u003e\u003cspan\u003e\\(\\chi\\)\u003c/span\u003e\u003c/span\u003e\u003csup\u003e2\u003c/sup\u003e = 7.293, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.0007), where +\u0026thinsp;G2 or \u0026ndash;G2 exhibited a greater relative change in root/shoot ratio than clone\u0026thinsp;+\u0026thinsp;G1 or -G1 (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). However, the endosymbiont-infection status (\u003cspan\u003e\u003cspan\u003e\\(\\chi\\)\u003c/span\u003e\u003c/span\u003e\u003csup\u003e2\u003c/sup\u003e = 1.660, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.197) nor the genotype x endosymbiont interaction (\u003cspan\u003e\u003cspan\u003e\\(\\chi\\)\u003c/span\u003e\u003c/span\u003e\u003csup\u003e2\u003c/sup\u003e = 0.320, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.571) did not show a significant effect in the GLMMs.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn our study, we evaluated the role of the facultative endosymbiont of aphids \u003cem\u003eR. insecticola\u003c/em\u003e on intra and interclonal competitive interactions of aphids. Our experiments revealed that, regardless the presence of \u003cem\u003eR. insecticola\u003c/em\u003e, in the cases of single colonies the clone G1 exhibited a higher PGR than clone G2. However, in competitive interclonal coexistence, G2 outperformed clone G1. These results suggest that aphid clones have differential features that allow more efficient performance as coexisting or solitary colonies, but not in both contexts. Surprisingly, clone G2 exhibited a higher PGR when coexisting with G1 than when developing alone or intraclonal coexistence. Variation in competitive abilities among aphid clones has been observed among clones for other aphid species such as the pea aphid, \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e (Hazell et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), but not including the effect of facultative endosymbionts. Similar results were also observed between a mutant colour (yellow) and its original (green) aphid clone which regulated its reproductive rate depending on the relative density of self and non-self-clones, but in this study only one aphid genotype was studied (Li and Akimoto \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which involved only intraclonal competition. In this study, authors have suggested that aphid clones exposed to clonal coexistence and single rearing could alter their reproduction by discriminating between closely related and unrelated clones in mixed colonies. In Chile, \u003cem\u003eS. avenae\u003c/em\u003e aphid shows strong signatures of obligated parthenogenesis and aphid clones are frequently observe over several seasons (Figueroa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Interestingly, the clone G1 and G2 of \u003cem\u003eS. avenae\u003c/em\u003e used in our study were found to be common in wheat and oat plantations of centre-south of Chile (G1 and G4 respectively in Zepeda-Paulo et al. (2021). In the field, the clone G1 has been observed as the dominant clone at the beginning of the season compared to the other clones (including G2), but this decreased from the mid-season, on the contrary the clone G2 increased its prevalence throughout the season being dominant at the end of the season (Zepeda-Paulo et al. 2021). In light of our results, temporal dynamic and dominance of aphid clones throughout the growing season could be explained in part by a differential response of aphid clones, by example when they are exposed to lesser competitive (e.g., early season) and highly competitive scenarios (i.e., growing season), which would contribute to maintaining clonal variation within natural populations. The greater competitive ability of the clone G2 can be explained by a rapid occupation of the resource, developing a niche pre-emption strategy. This result supports Silvertown`s hypothesis (2004) for niche pre-emption limiting the diversification of late-arriving lineages. Evidence supporting this is the higher number of individuals of clone G2 than G1 at day 15 developing with or without harbouring \u003cem\u003eR. insecticola\u003c/em\u003e, with clone G2 reaching an asymptote at a stage when clone G1 growing alone was initiating an exponential type of growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsistent with our data, a similar study found that one clone of aphid species \u003cem\u003eMyzus persicae\u003c/em\u003e outgrew another clone only under coexistence (Turcotte et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Thus, aphid clones appear interact when mixed, leading to an asymmetrical competitive interaction, which in turn can drive eco-evolutionary feedback loops. However, in that work Turcotte et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003eb\u003c/span\u003e) did not explore the mechanism underpinning clonal interaction. In our study, unlike our initial prediction, we can state that the asymmetric interaction between clone G1 and G2 appear not to be related with the presence of endosymbionts. Although competitive interactions between aphid clones was not significantly influenced by endosymbiont-infection, we have found that individual of \u003cem\u003eS. avenae\u003c/em\u003e aphids harbouring the facultative endosymbiont \u003cem\u003eR. insecticola\u003c/em\u003e exhibited a greater PGR than endosymbiont-free aphids. Furthermore, the positive effect on PGR that \u003cem\u003eR. insecticola\u003c/em\u003e inflicts on the different aphid clones studied here agrees with the fact that positive selection of aphid clones harbouring \u003cem\u003eR. insecticola\u003c/em\u003e in \u003cem\u003eS. avenae\u003c/em\u003e natural populations (Zepeda-Paulo et al. 2021). Since high \u003cem\u003eRegiella\u003c/em\u003e-infection levels were reached (87%-93%) over the course of a season on different cereal plants (wheat and oat) (Zepeda-Paulo et al. 2021), which shed some light on how facultative endosymbionts could drive different eco-evolutionary pathways of aphid clones at field level.\u003c/p\u003e \u003cp\u003eFor instance, the fact the clone G1 harbouring endosymbiont (+ G1) showed a higher fresh weight growing as solitary clone than -G1 and also than + G2 and -G2, suggest that \u003cem\u003eR. insecticola\u003c/em\u003e positively affects the traits of this clone. This positive effect could be associated with an adaptation to host plants, similar to when the reproduction of infected pea aphids on clover is enhanced (Tsuchida et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), as a defensive effect of \u003cem\u003eRegiella\u003c/em\u003e in the aphid \u003cem\u003eS. avenae\u003c/em\u003e has already been ruled out (Zepeda-Paulo et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eAnother interesting result is the effect of endosymbionts on the proportion of winged morphs in aphid clones exposed to intraclonal interactions. Herein we found that harbouring \u003cem\u003eR. insecticola\u003c/em\u003e affects the proportion of winged individuals in a clone-dependent manner. While \u003cem\u003eR. insecticola\u003c/em\u003e resulted in a lower final proportion of winged individuals in clone G1, this response changed when aphids were exposed to interclonal interactions, where clone G2 produced a higher proportion of winged morphs in response to clonal competence. Previous studies have confirmed that main factors inducing winged individuals in aphids are overcrowding and the presence of predation risk (Mehrparvar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and parasitism (Braendle et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). There is also genetic variation between clones in the winged morphs (Braendle et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Mixed effects on the production of winged individuals due to the presence of \u003cem\u003eR. insecticola\u003c/em\u003e have been described in the aphid pea, with evidence that support positive (Leonardo and Mondor \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and negative (Reyes et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), differences that have been attributed in part to the \u003cem\u003eRegiella\u003c/em\u003e genotype (Reyes et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A reduced production of winged individuals in aphids harbouring \u003cem\u003eR. insecticola\u003c/em\u003e in a temperature-dependent manner was also described in \u003cem\u003eS. avenea\u003c/em\u003e (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Here we add that variation in winged production in aphids harbouring facultative endosymbionts also appears to depend on coexistence with other clones.\u003c/p\u003e \u003cp\u003eWe also inquired about the impact of the study clones, either with or without harbouring \u003cem\u003eR. insecticola\u003c/em\u003e, on the relative change of root/shoot ratio in the host plant as a measured of plant stress induced by aphids. We observed that aphid infestation significantly increased the root/shoot ratio in wheat plants compared aphid-free plants. Feeding by aphids can affect plant growth and structure, mainly by reducing root tissue density, likely by a carbohydrate depletion in plants due to translocation from roots to shoots (Smith and Schowalter \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Interestingly, our results showed differences in the impact of aphids on the root/shoot ratio between aphid clones, indicating different levels of damage produced by the aphid clones. For instance, clone G1 showed a greater damaging capacity on host plants compared to clone G2, which could indicate for the aphid genotypes studied that clonal competition could be beneficial for plants as clone G2 outperformed clone G1 in competitive interaction. Although \u003cem\u003eR. insecticola\u003c/em\u003e appears to do not induce significant changes on the root/shoot ratio, aphids harbouring \u003cem\u003eR. insecticola\u003c/em\u003e showed a lesser damaging capacity on the host plants than endosymbiont-free aphids, which was true for both aphid clones studied. A similar tendency to show a lower root/shoot ratio in plants infected with aphids that harbour facultative endosymbionts was reported in the pea aphid-\u003cem\u003eHamiltonella defensa\u003c/em\u003e-broad bean system (Serteyn et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further studies are needed to understand how variation in the aphid genotype x endosymbiont interaction in different ecological contexts (e.g., intra and interclonal competition) affect growth, photosynthesis, and reproduction of host plants. .\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eThe results obtained in this study showed that aphid clones varied considerably in their population growth rate when they are exposed to competitive and unrivalled environments. That trade-off in competitive interactions among aphid clones can influence the dynamics of aphid populations and impact on plant growth and structure. While facultative endosymbionts like \u003cem\u003eR. insecticola\u003c/em\u003e do not play a significant role in directly mediating the competitive interactions of aphid clones or effecting specific plant traits, their presence does enhance aphid performance. Aphids harbouring \u003cem\u003eR. insecticola\u003c/em\u003e showed higher growth rates in various coexistence scenarios and across different clones with a concurrently reduced capacity to damage host plants.\u003c/p\u003e\u003cp\u003eThese findings are particularly relevant in the context of pest management in cereal crops. Understanding the role of facultative endosymbionts in aphid dynamics offers new perspectives for developing more specific and sustainable pest control strategies. Leveraging the differential effects of R. \u003cem\u003einsecticola\u003c/em\u003e on aphid competition and plant health could lead to more effective and ecologically conscious approaches for managing aphid populations in agricultural settings. Ultimately, our study underscores the importance of considering symbiotic relationships in pest management and contributes to a more nuanced understanding of the eco-evolutionary dynamics of pest species in agroecosystems. This ecological perspective is vital not only for predicting changes in pest populations but also for developing sustainable and environmentally friendly control methods.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.R. and M.M. Conceptualization; C.R. and M.M. Methodology; F.Z. and M.M. data collection; M.R. molecular analyses; M.M. and F.Z. formal data analysis; M.M. and M.R. writing - original draft preparation; C.R. and F.Z. writing\u0026mdash;review and editing; C.R. and F.Z. funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBorodovsky M, McIninch J (1993) GENMARK: parallel gene recognition for both DNA strands. Computers and Chemistry 17(2):123-133 https://doi.org/10.1016/0097-8485(93)85004-V\u003c/li\u003e\n\u003cli\u003eBraendle, C., Friebe, I., Caillaud, M. C., and Stern, D. L. (2005). Genetic variation for an aphid wing polyphenism is genetically linked to a naturally occurring wing polymorphism. Proceedings of the Royal Society B: Biological Sciences 272(1563): 657\u0026ndash;664 https://doi.org/10.1098/rspb.2004.2995\u003c/li\u003e\n\u003cli\u003eBraendle C, Davis G K, Brisson J A, Stern DL (2006) Wing dimorphism in aphids. Heredity 97(3): 192-199 https://doi.org/10.1038/sj.hdy.6800863\u003c/li\u003e\n\u003cli\u003eBruijning M, Jongejans E, Turcotte MM (2019) Demographic responses underlying eco‐evolutionary dynamics as revealed with inverse modelling. Journal of Animal Ecology 88(5):768-779 https://doi.org/10.1111/1365-2656.12966\u003c/li\u003e\n\u003cli\u003eDouglas AE (2009). The microbial dimension in insect nutritional ecology. Functional Ecology 23(1):38-47 https://doi.org/10.1111/j.1365-2435.2008.01442.x\u003c/li\u003e\n\u003cli\u003eFerrari J, Vavre F (2011) Bacterial symbionts in insects or the story of communities affecting communities. Philosophical Transactions of the Royal Society of London B: Biological Sciences 366(1569): 1389\u0026ndash;1400 https://doi.org/10.1098/rstb.2010.0226\u003c/li\u003e\n\u003cli\u003eFeldhaar H (2011) Bacterial symbionts as mediators of ecologically important traits of insect hosts. Ecological Entomology 36:533-543 https://doi.org/10.1111/j.1365-2311.2011.01318.x\u003c/li\u003e\n\u003cli\u003eFigueroa CC, Simon JG, Le Gallic JF, Prunier-Leterme N, Briones LM, Dedryver CA, Niemeyer HM (2005) Genetic structure and clonal diversity of an introduced pest in Chile, the cereal aphid \u003cem\u003eSitobion avenae.\u003c/em\u003e Heredity 95(1):24\u0026ndash;33 https://doi.org/10.1038/sj.hdy.6800662\u003c/li\u003e\n\u003cli\u003eFox J, Weisberg S, Adler D, Bates D, Baud-Bovy G, Ellison S, ... Heiberger R (2012) Package \u0026lsquo;car\u0026rsquo;. Vienna: R Foundation for Statistical Computing, 16.\u003c/li\u003e\n\u003cli\u003eHazell SP, Gwynn DM, Ceccarelli S, Fellowes MDE (2005) Competition and dispersal in the pea aphid: clonal variation and correlations across traits. Ecological Entomology 30(3):293-298 https://doi.org/10.1111/j.0307-6946.2005.00703.x\u003c/li\u003e\n\u003cli\u003eHertaeg C, Risse M, Vorburger C, De Moraes CM, Mescher MC (2021) Aphids harbouring different endosymbionts exhibit differences in cuticular hydrocarbon profiles that can be recognized by ant mutualists. Scientific Reports 11(1):19559. https://doi.org/10.1038/s41598-021-98098-2\u003c/li\u003e\n\u003cli\u003eHothorn T, Bretz F, Westfall P, Heiberger RM, Schuetzenmeister A, Scheibe S, Hothorn M T (2016) Package \u0026lsquo;multcomp\u0026rsquo;. Simultaneous inference in general parametric models. Project for Statistical Computing, Vienna, Austria.\u003c/li\u003e\n\u003cli\u003eKoga R, Tsuchida T, Fukatsu T (2003) Changing partners in an obligate symbiosis: a facultative endosymbiont can compensate for loss of the essential endosymbiont \u003cem\u003eBuchnera\u003c/em\u003e in an aphid. Proceedings of the Royal Society B: Biological Sciences, 270(1533), 2543\u0026ndash;2550. https://doi.org/10.1098/rspb.2003.2537\u003c/li\u003e\n\u003cli\u003eKoga R, Tsuchida T, Sakurai M, Fukatsu T (2007) Selective elimination of aphid endosymbionts: effects of antibiotic dose and host genotype, and fitness consequences. FEMS Microbiology Ecology, 60(2), 229\u0026ndash;239. https://doi.org/10.1111/j.1574-6941.2007.00284.x\u003c/li\u003e\n\u003cli\u003eLeigh SG, Emden HF van (2017) Population dynamics: cycles and patterns., CABI Books. CABI. https://doi.org/10.1079/9781780647098.0262.\u003c/li\u003e\n\u003cli\u003eLeonardo TE, Mondor EB (2006) Symbiont modifies host life-history traits that affect gene flow. Proceedings of the Royal Society B: Biological Sciences 273(1590):1079-84. https://doi/10.1098/rspb.2005.3408 \u003c/li\u003e\n\u003cli\u003eLi Y, Akimoto S I (2021) Self and non-self recognition affects clonal reproduction and competition in the pea aphid. Proceedings of the Royal Society B: Biological Sciences 288(1953): 20210787. https://doi.org/10.1098/rspb.2021.0787\u003c/li\u003e\n\u003cli\u003eLiu XD, Lei H X, Chen FF (2019) Infection pattern and negative effects of a facultative endosymbiont on its insect host are environment-dependent. Scientific Reports 9: 4013. https://doi/10.1038/s41598-019-40607-5\u003c/li\u003e\n\u003cli\u003eLlewellyn KS, Loxdale HD, Harrington R, Clark SJ, Sunnucks P (2004) Evidence for gene flow and local clonal selection in field populations of the grain aphid (\u003cem\u003eSitobion avenae\u003c/em\u003e) in Britain revealed using microsatellites. Heredity 93(2): 143-153. https://doi.org/10.1038/sj.hdy.6800466\u003c/li\u003e\n\u003cli\u003eLuo C, Chai R, Liu X, Dong Y, Desneux N, Feng Y, Hu Z (2022) The facultative \u003cem\u003esymbiont Regiella insecticola\u003c/em\u003e modulates non-consumptive and consumptive effects of \u003cem\u003eHarmonia axyridis\u003c/em\u003e on host aphids. Entomologia Generalis 42: 733-741. https://doi.org/10.1127/entomologia/2022/1368\u003c/li\u003e\n\u003cli\u003eŁukasik, P.; Dawid, M.A.; Ferrari, J.; Godfray, H.C.J. (2013) The diversity and fitness effects of infection with facultative endosymbionts in the grain aphid, \u003cem\u003eSitobion avenae\u003c/em\u003e. Oecologia 173: 985\u0026ndash;996. \u003c/li\u003e\n\u003cli\u003eMcFall-Ngai M, Hadfield MG, Bosch T CG, Carey HV, Domazet-Lo\u0026scaron;o T, Douglas AE, et al. (2013) Animals in a bacterial world, a new imperative for the life sciences. Proceedings of the National Academy of Sciences of the United States of America 110(9): 3229-3236. https://doi.org/10.1073/pnas.1218525110\u003c/li\u003e\n\u003cli\u003eMehrparvar M, Zytynska SE, Weisser W W (2013) Multiple cues for winged morph production in an aphid metacommunity. PLoS ONE 8(3): e58323. https://doi.org/10.1371/journal.pone.0058323\u003c/li\u003e\n\u003cli\u003eMoran NA (2006) Symbiosis. Current Biology 16(20): R866 - R871\u003c/li\u003e\n\u003cli\u003eOliver KM, Russell JA, Moran NA, Hunter M S (2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proceedings of the National Academy of Sciences 100(4):1803-1807. https:// doi.org/10.1073/pnas.0335320100. \u003c/li\u003e\n\u003cli\u003eOliver K M, Degnan P H, Burke GR, Moran N A (2010) Facultative symbionts in aphids and the horizontal transfer of ecologically important traits. Annual Review of Entomology 55: 247\u0026ndash;266. https://doi.org/10.1146/annurev-ento-112408-085305\u003c/li\u003e\n\u003cli\u003eOliver K M, Campos J, Moran N A, Hunter M S (2008) Population dynamics of defensive symbionts in aphids. Proceedings of the Royal Society of London B: Biological Sciences 275(1632): 293\u0026ndash;299. https://doi.org/10.1098/rspb.2007.1192\u003c/li\u003e\n\u003cli\u003eOliver KM, 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(4): 1803\u0026ndash;1807. https://doi.org/10.1073/pnas.0335320100\u003c/li\u003e\n\u003cli\u003ePeccoud J, Bonhomme J, Mah\u0026eacute;o F, de la Huerta M, Cosson O, Simon JG (2014) Inheritance patterns of secondary symbionts during sexual reproduction of pea aphid biotypes. Insect Science 21(3): 291\u0026ndash;300. https://doi.org/10.1111/1744-7917.12083\u003c/li\u003e\n\u003cli\u003ePetermann JS, M\u0026uuml;ller C B, Roscher C, Weigelt A, Weisser W W, Schmid B (2010) Plant species loss affects life-history traits of aphids and their parasitoids. PLoS ONE 5(8):e12053. https://doi.org/10.1371/journal.pone.0012053\u003c/li\u003e\n\u003cli\u003ePons I, Renoz F, Hance T (2019) Fitness costs of the cultivable symbiont \u003cem\u003eSerratia symbiotica\u003c/em\u003e and its phenotypic consequences to aphids in presence of environmental stressors. Evolutionary Ecology 33: 825-838.\u003c/li\u003e\n\u003cli\u003ePons I, Renoz F, No\u0026euml;l C, Hance T (2019b) Circulation of the cultivable symbiont \u003cem\u003eSerratia symbiotica\u003c/em\u003e in aphids is mediated by plants. Frontiers in Microbiology 10: 764. https://doi.org/10.3389/fmicb.2019.00764\u003c/li\u003e\n\u003cli\u003eKaech H, Jud S, Vorburger C (2022) Similar cost of Hamiltonella defensa in experimental and natural aphid‐endosymbiont associations. Ecology and Evolution 12(1): e8551. https://doi.org/10.1002/ece3.8551\u003c/li\u003e\n\u003cli\u003eR Core Team (2018) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-C\u0026aacute;ceres G E, Moya-Hern\u0026aacute;ndez M G, Quilodr\u0026aacute;n M, Nespolo R F, Ceballos R, Villagra CA Ram\u0026iacute;rez, C C (2019) Harbouring the secondary endosymbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e increases predation risk and reproduction in the cereal aphid \u003cem\u003eSitobion avenae\u003c/em\u003e. Journal of Pest Science 92: 1039-1047. https://doi.org/10.1007/s10340-019-01090-z\u003c/li\u003e\n\u003cli\u003eReyes M L, Laughton A M, Parker B J, et al. (2019) The influence of symbiotic bacteria on reproductive strategies and wing polyphenism in pea aphids responding to stress. Journal of Animal Ecology 88: 601\u0026ndash;611. https://doi.org.utalca.idm.oclc.org/10.1111/1365-2656.12942\u003c/li\u003e\n\u003cli\u003eScarborough CL, Ferrari J, Godfray H C J (2005) Aphid protected from pathogen by endosymbiont. Science 310(5755): 1781. https://doi.org/10.1126/science.1120180\u003c/li\u003e\n\u003cli\u003eSchillewaert S, Parmentier T, Vantaux A, Van den Ende W, Vorburger C, Wenseleers T (2017) The influence of facultative endosymbionts on honeydew carbohydrate and amino acid composition of the black bean aphid Aphis fabae. Physiological Entomology 42(2): 125-133. https://doi.org/10.1111/phen.12181\u003c/li\u003e\n\u003cli\u003eSerteyn L, Quaghebeur C, Ongena M, Cabrera N, Barrera A, Molina-Montenegro MA, Francis F, Ram\u0026iacute;rez CC (2020) Induced systemic resistance by a plant growth-promoting rhizobacterium impacts development and feeding behavior of aphids. Insects 11(4):234. https://doi.org/10.3390/insects11040234\u003c/li\u003e\n\u003cli\u003eSchuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18(2): 233-234. https://doi.org/10.1038/72708\u003c/li\u003e\n\u003cli\u003eSimon J C, Baumann S, Sunnucks P, Hebert PD N, Pierre JS, Le Gallic JF, Dedryver C A (1999) Reproductive mode and population genetic structure of the cereal aphid \u003cem\u003eSitobion avenae\u003c/em\u003e studied using phenotypic and microsatellite markers. Molecular Ecology 8(4): 531-545. https://doi.org/10.1046/j.1365-294x.1999.00583.x\u003c/li\u003e\n\u003cli\u003eSimon JG, Boutin S, Tsuchida T, Koga R, Le Gallic JF, Frantz A, Fukatsu T (2011) Facultative symbiont infections affect aphid reproduction. PLoS ONE 6(7): e21831. https://doi.org/10.1371/journal.pone.0021831\u003c/li\u003e\n\u003cli\u003eSmith J P, Schowalter TD (2001) Aphid‐induced reduction of shoot and root growth in Douglas‐fir seedlings. Ecological Entomology 26(4): 411-416.\u003c/li\u003e\n\u003cli\u003eSudakaran S, Kost C, Kaltenpoth M (2017) Symbiont acquisition and replacement as a source of ecological innovation. Trends in Microbiology, 25(5), 375-390. https://doi:org/10.1016/j.tim.2017.02.014. \u003c/li\u003e\n\u003cli\u003eSunnucks P, Hales D F (1996) Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae). Molecular Biology and Evolution 13(3): 510-524. https://doi.org/10.1093/oxfordjournals.molbev.a025612\u003c/li\u003e\n\u003cli\u003eThomas S, Dogimont C, Boissot N (2011) Association between Aphis gossypii genotype and phenotype on melon accessions. Arthropod-Plant Interactions 6(1): 93\u0026ndash;101. https://doi.org/10.1007/s11829-011-9155-2\u003c/li\u003e\n\u003cli\u003eTsuchida T, Koga R, Fukatsu T (2004) Host plant specialization governed by facultative symbiont. Science 303(5666): 1989. https://doi.org/10.1126/science.1094611\u003c/li\u003e\n\u003cli\u003eTsuchida T, Koga R, Horikawa M, Tsunoda T, Maoka T, Matsumoto S, Simon JG, Fukatsu T (2010) Symbiotic bacterium modifies aphid body color. Science 330(6007): 1102-1104. https://doi.org/10.1126/science.1195463\u003c/li\u003e\n\u003cli\u003eTurcotte M M, Reznick DN, Hare J D (2011a) Experimental assessment of the impact of rapid evolution on population dynamics. Evolutionary Ecology Research 13(2): 113\u0026ndash;131. https://doi.org/10.1111/j.1461-0248.2011.01676.x\u003c/li\u003e\n\u003cli\u003eTurcotte MM, Reznick D N, Hare J D (2011b) The impact of rapid evolution on population dynamics in the wild: experimental test of eco-evolutionary dynamics. Ecology Letters 14(11): 1084\u0026ndash;1092. https://doi.org/10.1111/j.1461-0248.2011.01676.x\u003c/li\u003e\n\u003cli\u003eVorburger C, Perlman S J (2018) The role of defensive symbionts in host\u0026ndash;parasite coevolution. Biological Review 93:1747-1764. https://doi.org/10.1111/brv.12417\u003c/li\u003e\n\u003cli\u003eWagner SM, Martinez A J, Ruan YM, Kim K L, Lenhart P A, Dehnel A C, Oliver KM, White J A (2015) Facultative endosymbionts mediate dietary breadth in a polyphagous herbivore. Functional Ecology 29(11): 1402\u0026ndash;1410. https://doi.org/10.1111/1365-2435.12459\u003c/li\u003e\n\u003cli\u003eWilson A C C, Massonnet B, Simon JG, Prunier-Leterme N, Dolatti L, Llewellyn K S, Figueroa C C, Ram\u0026iacute;rez C C, Blackman R L, Estoup A, Sunnucks P (2004) Cross-species amplification of microsatellite loci in aphids: assessment and application. Molecular Ecology Notes 4(1): 104\u0026ndash;109. https://doi.org/10.1046/j.1471-8286.2004.00584.x\u003c/li\u003e\n\u003cli\u003eWood S (2018) Mixed GAM computation vehicle with automatic smoothness estimation. R package version 1.8-24.\u003c/li\u003e\n\u003cli\u003eXin J-J, Shang Q-L, Desneux N, Gao X-W (2014) Genetic diversity of \u003cem\u003eSitobion avenae\u003c/em\u003e (Homoptera: Aphididae) populations from different geographic regions in China. PLoS ONE 9(10): e109349. https://doi.org/10.1371/journal.pone.0109349\u003c/li\u003e\n\u003cli\u003eZepeda-Paulo F, Lavandero B (2021) Effect of the genotypic variation of an aphid host on the endosymbiont associations in natural host populations. Insects 12(3): 217. https://doi.org/10.3390/insects12030217\u003c/li\u003e\n\u003cli\u003eZepeda‐Paulo FA, Villegas C, Lavandero B (2017) Host genotype\u0026ndash;endosymbiont associations and their relationship with aphid parasitism at the field level. Ecological Entomology 42(1): 86-95. https://doi.org/10.1111/een.12361\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"interclonal, intraclonal interactions, performance, winged morph, aphid, endosymbionts, Regiella insecticola","lastPublishedDoi":"10.21203/rs.3.rs-4021194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4021194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial endosymbionts are key components of aphid biology, as they modify several traits of their insect hosts. Here we studied how bacterial facultative endosymbionts affect the competitive interactions between aphid clones. To address this, we studied intraclonal and interclonal interactions between the two most common clones (G1 and G2) of the cereal aphid \u003cem\u003eSitobion avenae\u003c/em\u003e (Fabricius), including the role of the facultative endosymbiont \u003cem\u003eRegiella insecticola\u003c/em\u003e in the outcome of these interactions in a shared host (wheat). The results of this study reveal significant variability in the population growth rates of aphid clones under competitive and non-competitive environments. That trade-off in competitive interactions among aphid clones can influence the dynamics of aphid populations and impact on plant growth and structure. While facultative endosymbionts like \u003cem\u003eR. insecticola\u003c/em\u003e do not play a significant role in directly mediating the competitive interactions of aphid clones or affecting specific plant traits, their presence does enhance aphid performance. Aphids harbouring \u003cem\u003eR. insecticola\u003c/em\u003e showed higher growth rates in various coexistence scenarios and across different clones with a concurrently reduced capacity to damage host plants, which suggests that \u003cem\u003eR. insecticola\u003c/em\u003e produces ecologically relevant consequences for aphids in cereal fields.\u003c/p\u003e","manuscriptTitle":"Are competitive interactions between aphid clones mediated by facultative endosymbionts?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 13:02:23","doi":"10.21203/rs.3.rs-4021194/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea935dd5-4aef-47b8-bb35-40ac282df7e0","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-29T12:40:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-11 13:02:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4021194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4021194","identity":"rs-4021194","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}