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Symbiont-mediated protection against fungal pathogens in pea aphids: a 1 role for pathogen specificity? 2 3 Running title: Symbiont-mediated protection from fungi in aphids 4 5 6 7 8 Benjamin J. Parker,* Chelsea J. Spragg,§ Boran Altincicek‡, and Nicole M. Gerardo 9 10 Department of Biology, Emory University, O. Wayne Rollins Research Center, 1510 E. Clifton 11 Rd. N.E., Atlanta, GA, 30322, USA 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 ______________________ 30 31 *Corresponding author: 32 33 Benjamin Parker 34 Gerardo Lab 35 O. Wayne Rollins Research Center 36 1510 Clifton Road NE, Room 2006 37 Atlanta, GA 30322 38 39 Phone: +1 404 727 9344 40 Fax: +1 404 727 2880 41 42 [email protected] 43 44 45 46 §Present address: University of Washington, Program in Molecular and Cellular Biology 47 ‡Present address: University of Bonn, INRES-Phytomedicine, Germany48
Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.03193-12 AEM Accepts, published online ahead of print on 25 January 2013
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Abstract 49 50 Here we show that a bacterial endosymbiont, Regiella insecticola, protects pea aphids 51 (Acrythosiphon pisum) from the aphid-specific fungal entomopathogen Zoophthora 52 occidentalis, but not from the generalist insect fungal pathogen Beauveria bassiana. This 53 finding highlights the complex influence of fungi on the dynamics of this economically important 54 agricultural pest. 55 56
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Symbiotic relationships between invertebrates and vertically transmitted microbes are 57 widespread. One feature of this mutualistic relationship is that symbionts depend on host 58 resources for their own survival and reproduction (8), and theory therefore predicts that in the 59 absence of manipulation of host reproduction, beneficial symbionts must provide a fitness 60 advantage to spread through a host population (6). Studying ecologically relevant traits 61 conferred to hosts by symbionts is critical for understanding host-microbe dynamics, and 62 researchers have therefore searched for fitness advantages of harboring symbionts in a 63 number of systems. Several recent studies have shown that one advantage conferred by some 64 symbionts is protection against pathogens and parasites (13, 19, 27, 28, 31). For example, pea 65 aphids (Acyrthosiphon pisum), which are a model system for the study of host-symbiont 66 dynamics, are protected against the fungal entomopathogen Pandora neoaphidis 67 (Zygomycota: Entomophthorales) by several facultative, vertically transmitted bacteria, 68 including the gammaproteobacteria Regiella insecticola (15, 25). Fungi are important natural 69 pathogens of aphids (22) and are used in biocontrol (9, 23), and symbiont-mediated protection 70 to fungi is likely an important factor influencing the population dynamics of aphids and their 71 symbionts. However, aphids encounter several diverse species of fungal pathogens in the wild 72 (18). It is not known if Regiella-conferred protection is specific to Pandora or if it extends to 73 other species of fungus as well, which would suggest that multiple fungal species are 74 influencing aphid-Regiella dynamics. We therefore exposed pea aphids, with and without 75 symbionts, to two additional species of fungal pathogens: Zoophthora occidentalis 76 (Zygomycota: Entomophthorales), a highly aphid-specific entomopathogen, and Beauveria 77 bassiana (Ascomycota:: Hypocreales), a generalist that has been found in a variety of hosts, 78 including species of Coleoptera, Hemiptera, and Diptera (10, 18, 30). These fungal species are 79 highly divergent (with some estimates as high as 1000 mya (11)), but both species reproduce 80 by passively releasing spores (conidia) that penetrate the cuticle of a suitable host. Mycelia 81
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then colonize the host’s body tissue until the death of the host, when new spores are produced 82 and released into the environment. 83
We used two aphid genotypes, both with and without Regiella present (5A, collected in the 84 wild in 1999 near Madison, Wisconsin and subsequently injected with Regiella symbionts from 85 an aphid collected in Tompkins Co., NY, in 2000; and LSR1, collected on alfalfa near Ithaca, 86 NY in 1998 with a natural Regiella infection and then artificially cleared of symbionts.) (24). 87 The use of two aphid genotypes each with and without Regiella allowed us to control for 88 effects of aphid genotype on pathogen susceptibility. Before fungal infection, we maintained 89 pea aphids asexually on fava bean (Vicia faba) plants at 16hrs light: 8hrs dark at 20°C. We 90 exposed adults to fungus after their final molt (at nine days of age), as we found that molting 91 shortly after exposure strongly reduces infection probability. After exposure, aphids were kept 92 individually for 4 days on fava bean plants under near 100% humidity, after which the humidity 93 was reduced to 70%. This allowed enough time for fungal penetration of the aphid cuticle, 94 which requires high humidity. 95 Zoophthora (specialist) infection: We infected aphids with Zoophthora by placing them 96
under a ‘spore shower' (based on (3-5, 10, 25)). An isolate of Zoophthora was obtained from 97 the USDA ARS Collection of Entomopathogenic Fungal Cultures, and was grown for 2 weeks 98 on SDAEY plates at 20°C (20). Approximately 15 hours before infection, several small pieces 99 of fungal mycelium (3mm2) were cut with a sterile instrument and placed onto 1.5% tap water 100 agar, which causes the fungus to sporulate. At the time of infection, the agar plates were 101 inverted over a hollow tube for 60 minutes with aphids at the bottom of the chamber. The agar 102 plates were rotated among treatment groups during infection to ensure that each treatment 103 group was exposed to an equal dose of fungal spores (approximately 16.5 spores/mm2). We 104 included a glass slide in this rotation so that spores could be counted under a light microscope 105
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to estimate spore density. Control aphids were handled similarly but were not exposed to 106 fungus. 107
The Zoophthora infection was divided into three blocks conducted several days apart from 108 one another. In each block, we exposed two-thirds of the individuals from each genotype to 109 fungus, and kept one-third under identical conditions as a control. Half of the aphids of each 110 genotype harbored Regiella. Nine days after exposure, we recorded the survival of each aphid. 111 We fit a logistic regression model (a Generalized Linear Model (GLM) with quasibinomial error 112 structure and logit link function) to aphid survival with symbiont, fungal exposure, and block as 113 fixed effects (7). We used R version 2.11 for our statistical analyses. As expected, aphids 114 exposed to fungus had significantly lower survival than control aphids (odds ratio (OR) = 0.33, 115 95% confidence interval (CI) = [0.15, 0.72], p = 5.8*10-3). There was also a significant 116 interaction between exposure and symbiont-infection on aphid survival (OR = 3.7, CI = [1.2, 117 11], p = 0.021), but no effect of Regiella independently (OR = 0.70, CI = [0.28, 1.73], p = 0.44). 118 This means that in the absence of fungal infection Regiella did not have a significant impact on 119 aphid survival, but it increased survival of aphids exposed to fungus (figure 1). The trends were 120 consistent across three blocks, but aphid survival differed across blocks (block 2: OR = 2.6, CI 121 = [1.4, 4.7], p = 2.5*10-3 and block 3: OR = 3.4, CI = [1.8, 6.7], p = 3.2*10-4). The trend was 122 consistent across both aphid genotypes, and there was no significant effect of aphid genotype 123 on survival; it was therefore removed from the model. More aphid genotypes will need to be 124 tested to determine the effect, if any, of aphid genotype on fungal infection outcome. Using this 125 same protocol and these same genotypes, we also confirmed the results of Scarborough et al. 126 (2005)—that Regiella protects pea aphids from Pandora, which, like Zoophthora, is an aphid-127 specific fungal pathogen (Supplementary Figure A1). We also conducted a repeat of the 128 Zoophthora infection, using a second pathogen genotype, to ensure that our results were 129 consistent across multiple experiments (Supplementary Figure A2). 130
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Beauveria (generalist) infection: Cultures of Beauveria did not sporulate upon transfer to 131 tap water agar, so we instead made up solutions of Beauveria spores (Strain GHA, Botanigard 132 es) in distilled water. Spores were washed and separated via centrifuge from inert ingredients, 133 and 0.7μL of the solution was pipetted onto the dorsal side of the abdomen of each aphid. Half 134 of the aphids of each genotype harbored Regiella. We exposed aphids to four different spore 135 doses (0, 25, 250, and 2500 spores, estimated using a HYCOR KOVA Glasstic 136 hemocytometer). Aphids became infected with Beauveria faster than with Zoophthora, so for 137 the Beauveria infections we recorded the survival status of each aphid at 24hr intervals after 138 exposure, and we analyze this data using a survival analysis (figure 2). For the Beauveria 139 infection we took the additional precaution of transferring the symbiont from the 5A line into the 140 LSR1 aphid genotype. Symbiont free first instar LSR1 aphids were exposed to the hemolymph 141 of 5A adult aphids that harbored Regiella via intrahemocoellic microinjection. Aphids were then 142 kept for at least 10 generations to allow the symbiosis to stabilize and to allow the host lines to 143 adapt to the presence of the bacteria (14). This allowed us to compare survival of genetically 144 identical hosts with two different genotypes of symbionts. Survival data were analyzed using 145 age-specific parametric survival models with a Weibull distribution using the Survival package 146 in R version 2.11. The dose of fungal pathogen exposure had a significant impact on aphid 147 survival (minimal model containing spore dose only: χ2 = 935.96 on 3 d.f., p < 0.0001). 148 However, symbiont status had no significant effect on survival, had no interaction with dose, 149 and was removed from the minimal model. This suggests that Regiella did not protect aphids 150 from infection with Beauveria (figure 2). Aphid genotype had no significant effect on survival 151 and was also removed from the minimal model. We repeated this experiment with aphids that 152 harbored two other species of aphid secondary symbionts, H. defensa and S. symbiotica, and 153 again found no effect of symbionts on aphid susceptibility to Beauveria (Supplementary Figure 154 B1). We also conducted a repeat of the Beauveria infection using different spore doses and 155
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found no effect of Regiella (Supplementary Figure B2). Lastly, to ensure that this negative 156 result was not due to a sampling effect of Regiella genotypes, we conducted an additional 157 experiment where we collected aphids with and without Regiella from several geographical 158 locations, and assayed their resistance to Beauveria. With this experimental design, we are not 159 controlling for host genotype, as each strain used in the experiment will differ both in terms of 160 symbiont and host genotypic background. However, we are able to determine if multiple wild–161 collected lines with Regiella are, on average, more resistant to Beauveria than wild–collected 162 lines without symbionts. We found significant variation in resistance to Beauveria among aphid 163 genotypes, but no effect of harboring Regiella symbionts (see Supplementary information C), 164 suggesting that our results are not due to a lack of diversity in symbiont genotypes. 165
Together these data suggest that Regiella symbionts confer protection against several 166 specialist fungal pathogens, but not against a generalist pathogen. Regiella frequencies vary 167 among aphid populations (17, 29), and researchers have tried to determine the factors that 168 influence symbiont frequencies as a way of better understanding this model host-microbe 169 interaction. Variation in Regiella frequency is explained in part as a balance between the 170 benefits of protection from fungal pathogens and the costs of harboring bacteria. Our results 171 suggest that several species of fungal pathogens may be driving this interaction, but that 172 Regiella is not beneficial against all species of fungi. In addition, these fungal pathogens are 173 used in aphid biocontrol (9, 12, 16), and our results suggest that symbiont-mediated protection 174 against pathogens may be an important consideration when selecting and developing 175 biocontrol agents. 176
In general, researchers are working to develop an understanding of how evolution acts on 177 alternative defenses (like that conferred from symbionts) organisms have to protect 178 themselves from pathogens and parasites (21). One possible explanation for the pattern of 179 symbiont-mediated protection observed here is that Regiella protection has evolved in 180
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response to pressure from individual species of fungal pathogens. A second possibility is that 181 protection in this system has evolved in response to fungal pathogens that specialize on 182 aphids, but not to generalist insect pathogens, perhaps due to broad differences found in the 183 infection strategies of generalists vs. specialists (2, 26). Pathogen specificity has been shown 184 to be an important factor influencing host-enemy interactions (1), and we therefore highlight 185 the relative specificity of these pathogens as a potential explanation for the pattern of 186 protection observed in this system, but clearly more data are needed. It is possible that close 187 co-evolution between aphid specialists and Regiella is needed to develop or maintain 188 protection conferred by symbionts. 189
190 Thierry Lefèvre, Alice Laughton, Eleanore Sternberg, Anand Bhardwaj, and four anonymous 191 reviewers provided valuable assistance with the manuscript. We would like to especially thank 192 Richard Humber, Karen Hansen, and the USDA ARS Collection of Entomopathogenic Fungal 193 Cultures who provided cultures of Zoophthora. This research was supported by NSF IOS-194 1025853 to NMG. BA was supported by a DFG grant (AL902/4-1), and BJP is supported by an 195 NSF Graduate Research Fellowship. 196 197
198 199
Figure 1: Results of infection with Zoophthora. Survival of each aphid was recorded 9 days 200 after infection, and is reported as % survival of each group. White bars represent aphids with 201 no secondary symbionts (n = 138), and grey bars represent aphids that harbored Regiella (n = 202 131). Error bars are +/- standard error. 203 204 Figure 2: Results of infection with Beauveria. Survival of each aphid was recorded every 24 205 hours between 0 and 8 days after infection, and is reported as survival curves measured in % 206 survival of each group. A: Spore Dose. Each line represents a different spore dose (Control - 207 0 spores/aphid, Low - 25, Medium - 250, High - 2500), n = 175 per dose. B: Symbiont Status. 208 The solid line shows infected aphids without Regiella (n = 210). The dotted line shows infected 209 aphids that harbored Regiella (n = 315). 210
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