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Year: 2010

A strain of the bacterial symbiont Regiella insecticola protects against parasitoids

Vorburger, C ; Gehrer, L ; Rodriguez, P

Abstract: Aphids commonly harbour facultative bacterial and may benefit from their presence through increased resistance to parasitoids. This has been demonstrated for and Serratia symbiotica, while a third common , Regiella insecticola, did not provide such protection. However, this symbiont was recently detected in a highly resistant clone of the peach-potato , Myzus persicae, from Australia. To test if resistance was indeed conferred by the endosymbiont, we eliminated it from this clone with antibiotics, and we transferred it to two other clones of the same and one clone of a different aphid species (Aphis fabae). Exposing these lines to the parasitoid Aphidius colemani showed clearly that unlike other strains of this bacterium, this specific isolate of R. insecticola provides strong protection against parasitic wasps, suggesting that the ability to protect their host against natural enemies may evolve readily in multiple species of endosymbiotic .

DOI: https://doi.org/10.1098/rsbl.2009.0642

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-27606 Journal Article Accepted Version

Originally published at: Vorburger, C; Gehrer, L; Rodriguez, P (2010). A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids. Biology Letters, 6(1):109-111. DOI: https://doi.org/10.1098/rsbl.2009.0642 Page 1 of 13 Submitted to Biology Letters

1 2 3 4 1 A strain of the bacterial symbiont Regiella insecticola 5 6 7 2 8 protects aphids against parasitoids 9 10 3 11 12 13 4 Christoph Vorburger*, Lukas Gehrer & Paula Rodriguez 14 15 5 16 17 18 6 Institute of Zoology, University of Zürich, 8057 Zürich, Switzerland 19 20 7 *E-mail: [email protected] Review Only 21 22 23 8 24 25 9 Aphids commonly harbour facultative bacterial endosymbionts and may benefit 26 27 10 from their presence through increased resistance to parasitoids. This has been 28 29 30 11 demonstrated for Hamiltonella defensa and Serratia symbiotica, while a third 31 32 12 common endosymbiont, Regiella insecticola, did not provide such protection. 33 34 13 35 However, this symbiont was recently detected in a highly resistant clone of the 36 37 14 peach-potato aphid, Myzus persicae, from Australia. To test if resistance was indeed 38 39 15 conferred by the endosymbiont, we eliminated it from this clone with antibiotics, 40 41 16 42 and we transferred it to two other clones of the same and one clone of a different 43 44 17 aphid species (Aphis fabae). Exposing these lines to the parasitoid Aphidius colemani 45 46 18 showed clearly that unlike other strains of this bacterium, this specific isolate of R. 47 48 49 19 insecticola provides strong protection against parasitic wasps, suggesting that the 50 51 20 ability to protect their host against natural enemies may evolve readily in multiple 52 53 21 species of endosymbiotic bacteria. 54 55 56 22 57 58 23 Keywords: Aphis fabae; endosymbiont; Myzus persicae; parasitoid; Regiella insecticola; 59 60 24 resistance

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1 2 3 25 1. INTRODUCTION 4 5 6 26 Hymenopteran parasitoids are important natural enemies of aphids and may strongly 7 8 27 reduce their population growth (Schmidt et al. 2003). Despite this strong selection, there 9 10 28 11 is enormous clonal variation for susceptibility to parasitoids in natural populations of 12 13 29 aphids (Henter & Via 1995; Ferrari et al. 2001; von Burg et al. 2008; Vorburger et al. 14 15 30 2009). Some of this variation is explained by genetic differences among aphid clones 16 17 31 18 (von Burg et al. 2008; Vorburger et al. 2009), but most of the variation is due to 19 20 32 endosymbiotic bacteriaFor that some Review clones possess (Oliver Only et al. 2003). In addition to the 21 22 33 obligate or primary endosymbiont Buchnera aphidicola, which serves a nutritional 23 24 34 25 function (Douglas 1998), aphids may harbour a number of facultative or secondary 26 27 35 endosymbionts. The best studied are Hamiltonella defensa, Serratia symbiotica and 28 29 36 Regiella insecticola (Moran et al. 2005). They are faithfully transmitted from mother to 30 31 32 37 offspring and have remarkable phenotypic effects on their hosts, including protection 33 34 38 against natural enemies. Hamiltonella defensa and S. symbiotica have both been shown to 35 36 39 37 increase resistance to parasitoids (Oliver et al. 2003), which is due to their carrying a 38 39 40 toxin-encoding bacteriophage (APSE) that is responsible for the defence (Oliver et al. 40 41 41 2009). Regiella insecticola, on the other hand, increases resistance to a fungal pathogen 42 43 42 44 (Ferrari et al. 2004; Scarborough et al. 2005), but does not seem to protect against 45 46 43 parasitoids (Oliver et al. 2003; Vorburger et al. 2009), although a comparative study by 47 48 44 Ferrari et al. (2004) suggested an association between infection with R. insecticola and 49 50 51 45 increased resistance to the parasitoid Aphidius eadyi in pea aphids. 52 53 46 In a recent study on a collection of Australian clones of the peach-potato aphid, Myzus 54 55 47 persicae, von Burg et al. (2008) found one R. insecticola-infected clone to be entirely 56 57 58 48 resistant to two species of parasitoids. Yet with just a single, naturally infected clone it 59 60 49 was not possible to infer whether the high resistance was a genetic effect or conferred by

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1 2 3 50 the endosymbiont. Here we report a study in which we separated these effects by 4 5 6 51 experimentally infecting other aphid clones with the same isolate of R. insecticola and by 7 8 52 curing the naturally infected clone with antibiotics. The results show clearly that unlike 9 10 53 11 other strains of R. insecticola, this specific isolate strongly increases resistance to 12 13 54 parasitoids, while also having a positive effect on aphid body size. 14 15 55 16 17 56 18 2. MATERIAL AND METHODS 19 20 57 (a) Insects For Review Only 21 22 58 We worked with four pairs of aphid lines, each representing a different clone either with 23 24 59 25 or without the R. insecticola isolate that was suspected to provide defence against 26 27 60 parasitoids. Clone 5.15 is the resistant clone of M. persicae described in von Burg et al. 28 29 61 (2008). It was collected in 2003 at Bacchus Marsh, Australia, and naturally harboured R. 30 31 32 62 insecticola. Its infection with this symbiont was diagnosed by sequencing part of the 16S 33 34 63 ribosomal RNA gene (von Burg et al. 2008). The sequence is deposited in GenBank (no. 35 36 64 R- 37 EF596788). We cured this clone from R. insecticola to create line 5.15 . For this we 38 39 65 injected adult females with a solution of 0.2 mg/ml of Gentamicin. Their offspring (F1) 40 41 66 produced on the second day after injection were reared singly until they were adult and 42 43 67 44 started to reproduce. Then we sacrificed the F1 adults and tested for the presence of R. 45 46 68 insecticola by diagnostic PCR, using a primer pair specific to this endosymbiont 47 48 69 (Tsuchida et al. 2006). Offspring of females that tested negative were propagated further. 49 50 51 70 After all of these lines also tested negative in the F2 and F3 generation, we just retained 52 53 71 one line for further use. 54 55 72 To transfer R. insecticola from clone 5.15 into three previously uninfected aphid 56 57 58 73 clones, we used a microinjection protocol similar to the one described in Oliver et al. 59 60 74 (2003). The recipients included two clones of M. persicae (5.3 and 7.9, also collected at

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1 2 3 75 Bacchus Marsh in 2003) and one clone of the black bean aphid, Aphis fabae, collected at 4 5 6 76 St. Margrethen, Switzerland, in 2006 (clone A06-405, Vorburger et al. 2009). The latter 7 8 77 was used to test if any protective effect of this strain of R. insecticola would also be 9 10 78 11 expressed in other aphid species. Briefly, we anaesthetized aphids with CO2 and 12 13 79 punctured adults of the donor clone 5.15 to suck up the extruding hemolymph with a fine 14  15 80 glass needle attached to a microinjection pump (FemtoJet , Eppendorf). This hemolymph 16 17 18 81 was then injected into fourth instar nymphs of the receiver clones. The surviving 19 20 82 recipients were placedFor individually Review on plants and allowed Only to reproduce until they died. 21 22 83 We only retained the last few F1 offspring they produced and tested them for infection 23 24 25 84 with R. insecticola by diagnostic PCR after they had reproduced. Progeny of positive F1 26 27 85 were propagated further and tested again in the F2 and F3 generations. All lines retained 28 29 86 30 their acquired infection, so we reduced them again to one infected line per clone, labelled 31 R5.15 R5.15 R5.15 32 87 5.3 , 7.9 and A06-405 . 33 34 88 As parasitoid we used Aphidius colemani, a species that is commonly used in 35 36 89 37 biocontrol of pest aphids and capable of parasitising M. persicae as well as A. fabae. 38 39 90 After a single egg is laid into an aphid nymph, the parasitoid larva develops inside the 40 41 91 still active aphid. The host is only killed after completion of the larval development, when 42 43 44 92 the parasitoid pupates inside its dried remains, forming a characteristic 'mummy'. 45 46 93 47 48 94 49 (b) Experimental procedures 50 51 95 The basic assay to measure susceptibility to parasitoids followed Henter & Via (1995): 52 53 96 we exposed groups of aphid nymphs to parasitoids for a fixed period of time and 54 55 97 56 determined the proportion of individuals that were successfully parasitised. 57 58 98 Before the start of the experiment, we reconfirmed the infection status of our eight 59 60 99 lines by diagnostic PCR. We then split each line into ten sublines and placed them at

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1 2 3 100 random positions in ten different trays (randomized complete blocks). Sublines were 4 5 6 101 reared at 20°C and a 16 h photoperiod on caged seedlings of either radish (Raphanus 7 8 102 sativum) for M. persicae or broad bean (Vicia faba) for A. fabae. To avoid confounding 9 10 103 11 differences among lines with environmental maternal or grand-maternal effects carried 12 13 104 over from the stock culture, we propagated the sublines for two generations before testing 14 15 105 individuals of the third subline generation. To start this test generation, we transferred 16 17 106 18 five adult females from each subline to new plants to reproduce. We removed the adults 19 20 107 again after 24 h and weighedFor them Review before disposal on aOnly Mettler MX5 microbalance 21 22 108 (Mettler-Toledo, Greifensee, Switzerland) to obtain an estimate of body size. Two days 23 24 109 25 later, when offspring were 48-72 h old, all aphid nymphs on the plants were counted 26 27 110 (mean colony size = 32.3 ± 4.8 SD). From these counts we calculated the average number 28 29 111 of offspring produced per adult as an estimate of daily fecundity. Then we added a single 30 31 32 112 female parasitoid from our stock culture to each caged colony of aphid nymphs for 24 h. 33 34 113 Due to an unforeseen shortage of female wasps, we could only expose six blocks to 35 36 114 37 parasitoids on the same day. The remaining four blocks were exposed on the following 38 39 115 day, when more wasps had emerged in our stock colony. This entailed that aphid nymphs 40 41 116 in blocks 7-10 were on average 24 h older when attacked than nymphs in blocks 1-6. Any 42 43 117 44 additional variation this might have caused entered the block factor of our analyses. Ten 45 46 118 days after exposure to parasitoids, mummies were clearly visible and counted. 47 48 119 49 50 51 120 (c) Analyses 52 53 121 All statistical analyses were carried out in R 2.7.1 (R Development Core Team 2008). 54 55 122 The proportion of aphids exposed to wasps that were mummified served as our estimate 56 57 58 123 of susceptibility to parasitoids and was analysed using a generalised linear model with 59 60 124 logit link and – due to overdispersion – quasibinomial errors. We tested for the effects of

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1 2 3 125 R. insecticola × 4 block, infection with , clone and the infection clone interaction. Adult 5 6 126 body mass and daily fecundity were analysed with linear models testing for the same 7 8 127 effects. 9 10 128 11 12 13 129 3. RESULTS 14 15 130 Infection with R. insecticola had a highly significant effect on aphid susceptibility to the 16 17 18 131 parasitoid A. colemani (Table 1). The originally resistant clone 5.15 became susceptible 19 20 132 when cured from R. insecticolaFor , whereasReview the three susceptible Only clones became completely 21 22 133 or – in the case of clone A06-405 – almost completely resistant when transfected with this 23 24 25 134 endosymbiont (Fig. 1). The significant difference among the four aphid clones is largely 26 27 135 due to the one A. fabae clone being mummified at a higher rate than the three M. persicae 28 29 136 30 clones when uninfected with R. insecticola (Table 1, Fig. 1). The block effect was also 31 32 137 significant, but there was no significant infection × clone interaction, showing that R. 33 34 138 insecticola had a similar effect in different, even heterospecific genetic backgrounds. 35 36 37 139 Infection with R. insecticola had a positive effect on aphid adult mass, which also 38 39 140 differed significantly among the four aphid clones (Table 1, Fig. 2). However, these 40 41 141 42 differences did not translate into variation in daily fecundity, which was similar for all 43 44 142 clones and not affected by R. insecticola (Table 1, Fig. 2). 45 46 143 47 48 144 49 4. DISCUSSION 50 51 145 We show that an isolate of the endosymbiotic bacterium R. insecticola from an Australian 52 53 146 clone of M. persicae strongly increases aphid resistance to a parasitic wasp. Such effects 54 55 56 147 have been reported previously for two other aphid symbionts, H. defensa and – to a lesser 57 58 148 extent – S. symbiotica (Oliver et al. 2003). It appears that the ability to protect their host 59 60 149 against natural enemies evolves readily in multiple species of bacterial endosymbionts.

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1 2 3 150 This is fascinating, if not surprising, given that under faithful vertical transmission, the 4 5 6 151 evolutionary interests of host and symbiont are well aligned. Thus, R. insecticola should 7 8 152 be added to the list of endosymbionts capable of defending aphids against parasitoids, 9 10 153 11 even if most strains of this bacterium may not possess this ability (Oliver et al. 2003; 12 13 154 Vorburger et al. 2009). In H. defensa, variation in the level of defence that different 14 15 155 strains provide has been linked to the copy number of the toxin-encoding bacteriophage 16 17 156 18 APSE, which is required for the protective phenotype (Oliver et al. 2009). Whether the 19 20 157 same is true for R. insecticolaFor remains Review to be investigated. Only First PCR screens did not 21 22 158 provide any evidence for APSE in the protective strain of R. insecticola described here 23 24 159 25 (Nancy Moran, pers. comm.), but this does not exclude the possibility of other phage 26 27 160 variants being involved. 28 29 161 Considering the strong benefit provided by defensive endosymbionts, it is surprising 30 31 32 162 that they are not more common in aphid populations. Possibly, there are also costs of 33 34 163 harbouring such bacteria. Here we found no evidence for this assumption, as aphids were 35 36 164 37 somewhat heavier and equally fecund when infected with R. insecticola. However, a 38 39 165 study by Oliver et al. (2008) on H. defensa indicates that costs may only be expressed 40 41 166 under more realistic conditions. In the case of R. insecticola, we only have evidence for 42 43 167 44 induced costs of symbiont-conferred resistance, as individuals of the naturally infected 45 46 168 clone 5.15 of M. persicae suffer from a strongly reduced fecundity after successfully 47 48 169 resisting a parasitoid attack (Vorburger et al. 2008). This would at least reduce the 49 50 51 170 benefits of harbouring R. insecticola. 52 53 171 54 55 172 We thank D. Bopp for access to the microinjector, A. Gouskov for his help with aphid 56 57 58 173 rearing, and N. Moran and two reviewers for comments on the manuscript. This work was 59 60 174 supported by the Swiss National Science Foundation (grant 3100A0-109266 to CV).

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1 2 3 175 REFERENCES 4 5 6 176 7 8 177 Douglas, A. E. 1998 Nutritional interactions in insect-microbial symbioses: Aphids and 9 10 178 11 their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43, 17-37. (doi: 12 13 179 doi:10.1146/annurev.ento.43.1.17). 14 15 180 Ferrari, J., Müller, C. B., Kraaijeveld, A. R. & Godfray, H. C. J. 2001 Clonal variation 16 17 181 18 and covariation in aphid resistance to parasitoids and a pathogen. Evolution. 55, 19 20 182 1805-1814. (doi: For10.1111/j.0014-3820.2001.tb00829.x). Review Only 21 22 183 Ferrari, J., Darby, A. C., Daniell, T. J., Godfray, H. C. J. & Douglas, A. E. 2004 Linking 23 24 184 25 the bacterial community in pea aphids with host-plant use and natural enemy 26 27 185 resistance. Ecol. Entomol. 29, 60-65. 28 29 186 Henter, H. J. & Via, S. 1995 The potential for coevolution in a host-parasitoid system. I. 30 31 32 187 Genetic variation within an aphid population in susceptibility to a parasitic wasp. 33 34 188 Evolution. 49, 427-438. 35 36 189 37 Moran, N. A., Russell, J. A., Koga, R. & Fukatsu, T. 2005 Evolutionary relationships of 38 39 190 three new species of living as symbionts of aphids and other 40 41 191 insects. Appl. Environ. Microbiol. 71, 3302-3310. (doi: 10.1128/AEM.71.6.3302- 42 43 192 44 3310.2005). 45 46 193 Oliver, K. M., Russell, J. A., Moran, N. A. & Hunter, M. S. 2003 Facultative bacterial 47 48 194 symbionts in aphids confer resistance to parasitic wasps. Proc. Natl. Acad. Sci. U. S. 49 50 51 195 A. 100, 1803-1807. (doi: 10.1073/pnas.0335320100). 52 53 196 Oliver, K. M., Campos, J., Moran, N. A. & Hunter, M. S. 2008 Population dynamics of 54 55 197 defensive symbionts in aphids. Proc. R. Soc. Lond. B 275, 293-299. (doi: 56 57 58 198 10.1098/rspb.2007.1192). 59 60

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1 2 3 199 Oliver, K. M., Degnan, P. H., Hunter, M. S. & Moran, N. A. 2009 Bacteriophages encode 4 5 6 200 factors required for protection in a symbiotic mutualism. Science 325, 992-994. (doi: 7 8 201 10.1126/science.1174463). 9 10 202 11 R Development Core Team. 2008 R: a language and environment for statistical 12 13 203 computing. http://cran.r-project.org. 14 15 204 Scarborough, C. L., Ferrari, J. & Godfray, H. C. J. 2005 Aphid protected from pathogen 16 17 205 18 by endosymbiont. Science 310, 1781-1781. (doi: 10.1126/science.1120180). 19 20 206 Schmidt, M. H., Lauer,For A., Purtauf, Review T., Thies, C., Schaefer, Only M. & Tscharntke, T. 2003 21 22 207 Relative importance of predators and parasitoids for cereal aphid control. Proc. R. 23 24 208 25 Soc. Lond. B 270, 1905-1909. (doi: 10.1098/rspb.2003.2469). 26 27 209 Tsuchida, T., Koga, R., Sakurai, M. & Fukatsu, T. 2006 Facultative bacterial 28 29 210 endosymbionts of three aphid species, Aphis craccivora, Megoura crassicauda and 30 31 32 211 Acyrthosiphon pisum, sympatrically found on the same host plants. Appl. Entomol. 33 34 212 Zool. 41, 129-137. (doi: 10.1303/aez.2006.129). 35 36 213 37 von Burg, S., Ferrari, J., Müller, C. B. & Vorburger, C. 2008 Genetic variation and 38 39 214 covariation of susceptibility to parasitoids in the aphid Myzus persicae – no evidence 40 41 215 for trade-offs. Proc. R. Soc. Lond. B 275, 1089-1094. (doi: 10.1098/rspb.2008.0018). 42 43 216 44 Vorburger, C., Gouskov, A. & von Burg, S. 2008 Genetic covariation between 45 46 217 effectiveness and cost of defence in aphids. Biol. Lett. 4, 674-676. (doi: 47 48 218 doi:10.1098/rsbl.2008.0382). 49 50 51 219 Vorburger, C., Sandrock, C., Gouskov, A., Castañeda, L. E. & Ferrari, J. 2009 Genotypic 52 53 220 variation and the role of defensive endosymbionts in an all-parthenogenetic host- 54 55 221 parasitoid interaction. Evolution. 63, 1439-1450. (doi: 10.1111/j.1558- 56 57 58 222 5646.2009.00660.x). 59 60 223

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1 2 3 224 Figure captions 4 5 6 225 7 8 226 Fig. 1. Susceptibility of experimental lines of aphids to the parasitoid Aphidius colemani. 9 10 227 11 Each bar represents the mean of 10 assays. 12 13 228 14 15 229 Fig. 2. Adult mass (top) and daily fecundity (bottom) of four aphid clones in the presence 16 17 230 18 and absence of the bacterial endosymbiont Regiella insecticola. 19 20 231 For Review Only 21 22 232 Short title: A defensive strain of Regiella insecticola 23 24 233 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Table 1. Generalised linear model results for the proportion of aphids mummified and linear model results for adult mass and daily fecundity. 4 5 6 7 Proportion mummified Adult mass Daily fecundity 8 9 10 Source of variation d.f. Deviance F P MS F P MS F P 11 12 13 Block 9 For 126.16 3.396Review 0.002 0.008 Only 1.368 0.222 0.538 0.521 0.854 14 15 Infection with R. insecticola 1 316.87 76.776 < 0.001 0.038 6.435 0.014 0.002 0.002 0.969 16 17 18 Aphid clone 3 40.08 3.237 0.028 0.020 3.298 0.026 0.297 0.287 0.835 19 20 Infection × clone 3 3.03 0.244 0.865 0.005 0.865 0.464 0.630 0.610 0.611 21 22 Residual 63 226.24 0.006 1.032 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 11 45 http://mc.manuscriptcentral.com/bl 46 47 Submitted to Biology Letters Page 12 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Fig. 1. Susceptibility of experimental lines of aphids to the parasitoid Aphidius colemani. Each bar 41 represents the mean of 10 assays. 42 139x141mm (600 x 600 DPI) 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/bl Page 13 of 13 Submitted to Biology Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Fig. 2. Adult mass (top) and daily fecundity (bottom) of four aphid clones in the presence and 49 absence of the bacterial endosymbiont Regiella insecticola. 143x263mm (600 x 600 DPI) 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/bl