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Year: 2010
A strain of the bacterial symbiont Regiella insecticola
protects aphids against parasitoids
Vorburger, C; Gehrer, L; Rodriguez, P
Postprint available at:
http://dx.doi.org/10.5167/uzh27606
Publisher's version available at:
http://dx.doi.org/10.1098/rsbl.2009.0642
Posted at the Zurich Open Repository and Archive, University of Zurich
http://www.zora.uzh.ch
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):109111.
A strain of the bacterial symbiont Regiella insecticola protects
aphids against parasitoids
Abstract
Aphids commonly harbour facultative bacterial endosymbionts and may benefit from their presence
through increased resistance to parasitoids. This has been demonstrated for Hamiltonella defensa
and Serratia symbiotica, while a third common endosymbiont, Regiella insecticola, did not provide
such protection. However, this symbiont was recently detected in a highly resistant clone of the
peachpotato aphid, 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 bacteria.
Page 1 of 13 This is the accepted manuscript of the article. The definitive verion is available at
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4 1 A strain of the bacterial symbiont Regiella insecticola
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2 protects aphids against parasitoids
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13 4 Christoph Vorburger*, Lukas Gehrer & Paula Rodriguez
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18 6 Institute of Zoology, University of Zürich, 8057 Zürich, Switzerland
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20 7 *E-mail: christoph.vorburger@zool.uzh.ch
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25 9 Aphids commonly harbour facultative bacterial endosymbionts and may benefit
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30 11 demonstrated for Hamiltonella defensa and Serratia symbiotica, while a third
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32 12 common endosymbiont, Regiella insecticola, did not provide such protection.
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35 13 However, this symbiont was recently detected in a highly resistant clone of the
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37 14 peach-potato aphid, Myzus persicae, from Australia. To test if resistance was indeed
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15 conferred by the endosymbiont, we eliminated it from this clone with antibiotics,
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42 16 and we transferred it to two other clones of the same and one clone of a different
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44 17 aphid species (Aphis fabae). Exposing these lines to the parasitoid Aphidius colemani
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18 showed clearly that unlike other strains of this bacterium, this specific isolate of R.
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49 19 insecticola provides strong protection against parasitic wasps, suggesting that the
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51 20 ability to protect their host against natural enemies may evolve readily in multiple
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21 species of endosymbiotic bacteria.
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56 22
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58 23 Keywords: Aphis fabae; endosymbiont; Myzus persicae; parasitoid; Regiella insecticola;
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25 1. INTRODUCTION
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6 26 Hymenopteran parasitoids are important natural enemies of aphids and may strongly
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8 27 reduce their population growth (Schmidt et al. 2003). Despite this strong selection, there
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28 is enormous clonal variation for susceptibility to parasitoids in natural populations of
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13 29 aphids (Henter & Via 1995; Ferrari et al. 2001; von Burg et al. 2008; Vorburger et al.
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15 30 2009). Some of this variation is explained by genetic differences among aphid clones
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18 31 (von Burg et al. 2008; Vorburger et al. 2009), but most of the variation is due to
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20 32 endosymbiotic bacteria that some clones possess (Oliver et al. 2003). In addition to the
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22 33 obligate or primary endosymbiont Buchnera aphidicola, which serves a nutritional
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27 35 endosymbionts. The best studied are Hamiltonella defensa, Serratia symbiotica and
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32 37 offspring and have remarkable phenotypic effects on their hosts, including protection
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34 38 against natural enemies. Hamiltonella defensa and S. symbiotica have both been shown to
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37 39 increase resistance to parasitoids (Oliver et al. 2003), which is due to their carrying a
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39 40 toxin-encoding bacteriophage (APSE) that is responsible for the defence (Oliver et al.
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41 41 2009). Regiella insecticola, on the other hand, increases resistance to a fungal pathogen
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42 (Ferrari et al. 2004; Scarborough et al. 2005), but does not seem to protect against
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46 43 parasitoids (Oliver et al. 2003; Vorburger et al. 2009), although a comparative study by
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48 44 Ferrari et al. (2004) suggested an association between infection with R. insecticola and
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51 45 increased resistance to the parasitoid Aphidius eadyi in pea aphids.
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53 46 In a recent study on a collection of Australian clones of the peach-potato aphid, Myzus
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47 persicae, von Burg et al. (2008) found one R. insecticola-infected clone to be entirely
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58 48 resistant to two species of parasitoids. Yet with just a single, naturally infected clone it
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60 49 was not possible to infer whether the high resistance was a genetic effect or conferred by
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50 the endosymbiont. Here we report a study in which we separated these effects by
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6 51 experimentally infecting other aphid clones with the same isolate of R. insecticola and by
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8 52 curing the naturally infected clone with antibiotics. The results show clearly that unlike
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53 other strains of R. insecticola, this specific isolate strongly increases resistance to
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13 54 parasitoids, while also having a positive effect on aphid body size.
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18 56 2. MATERIAL AND METHODS
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20 57 (a) Insects
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22 58 We worked with four pairs of aphid lines, each representing a different clone either with
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27 60 parasitoids. Clone 5.15 is the resistant clone of M. persicae described in von Burg et al.
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32 62 insecticola. Its infection with this symbiont was diagnosed by sequencing part of the 16S
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34 63 ribosomal RNA gene (von Burg et al. 2008). The sequence is deposited in GenBank (no.
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37 64 EF596788). We cured this clone from R. insecticola to create line 5.15R-. For this we
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39 65 injected adult females with a solution of 0.2 mg/ml of Gentamicin. Their offspring (F1)
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41 66 produced on the second day after injection were reared singly until they were adult and
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67 started to reproduce. Then we sacrificed the F1 adults and tested for the presence of R.
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46 68 insecticola by diagnostic PCR, using a primer pair specific to this endosymbiont
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48 69 (Tsuchida et al. 2006). Offspring of females that tested negative were propagated further.
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51 70 After all of these lines also tested negative in the F2 and F3 generation, we just retained
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53 71 one line for further use.
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72 To transfer R. insecticola from clone 5.15 into three previously uninfected aphid
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58 73 clones, we used a microinjection protocol similar to the one described in Oliver et al.
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60 74 (2003). The recipients included two clones of M. persicae (5.3 and 7.9, also collected at
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75 Bacchus Marsh in 2003) and one clone of the black bean aphid, Aphis fabae, collected at
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6 76 St. Margrethen, Switzerland, in 2006 (clone A06-405, Vorburger et al. 2009). The latter
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8 77 was used to test if any protective effect of this strain of R. insecticola would also be
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78 expressed in other aphid species. Briefly, we anaesthetized aphids with CO2 and
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13 79 punctured adults of the donor clone 5.15 to suck up the extruding hemolymph with a fine
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15 80 glass needle attached to a microinjection pump (FemtoJet, Eppendorf). This hemolymph
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18 81 was then injected into fourth instar nymphs of the receiver clones. The surviving
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20 82 recipients were placed individually on plants and allowed to reproduce until they died.
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83 We only retained the last few F1 offspring they produced and tested them for infection
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25 84 with R. insecticola by diagnostic PCR after they had reproduced. Progeny of positive F1
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27 85 were propagated further and tested again in the F2 and F3 generations. All lines retained
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30 86 their acquired infection, so we reduced them again to one infected line per clone, labelled
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32 87 5.3R5.15, 7.9R5.15 and A06-405R5.15.
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34 88 As parasitoid we used Aphidius colemani, a species that is commonly used in
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37 89 biocontrol of pest aphids and capable of parasitising M. persicae as well as A. fabae.
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39 90 After a single egg is laid into an aphid nymph, the parasitoid larva develops inside the
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91 still active aphid. The host is only killed after completion of the larval development, when
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46 93
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49 94 (b) Experimental procedures
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51 95 The basic assay to measure susceptibility to parasitoids followed Henter & Via (1995):
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53 96 we exposed groups of aphid nymphs to parasitoids for a fixed period of time and
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56 97 determined the proportion of individuals that were successfully parasitised.
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58 98 Before the start of the experiment, we reconfirmed the infection status of our eight
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60 99 lines by diagnostic PCR. We then split each line into ten sublines and placed them at
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100 random positions in ten different trays (randomized complete blocks). Sublines were
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6 101 reared at 20°C and a 16 h photoperiod on caged seedlings of either radish (Raphanus
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8 102 sativum) for M. persicae or broad bean (Vicia faba) for A. fabae. To avoid confounding
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103 differences among lines with environmental maternal or grand-maternal effects carried
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13 104 over from the stock culture, we propagated the sublines for two generations before testing
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15 105 individuals of the third subline generation. To start this test generation, we transferred
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18 106 five adult females from each subline to new plants to reproduce. We removed the adults
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20 107 again after 24 h and weighed them before disposal on a Mettler MX5 microbalance
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22 108 (Mettler-Toledo, Greifensee, Switzerland) to obtain an estimate of body size. Two days
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25 109 later, when offspring were 48-72 h old, all aphid nymphs on the plants were counted
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27 110 (mean colony size = 32.3 ± 4.8 SD). From these counts we calculated the average number
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111 of offspring produced per adult as an estimate of daily fecundity. Then we added a single
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32 112 female parasitoid from our stock culture to each caged colony of aphid nymphs for 24 h.
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34 113 Due to an unforeseen shortage of female wasps, we could only expose six blocks to
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37 114 parasitoids on the same day. The remaining four blocks were exposed on the following
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39 115 day, when more wasps had emerged in our stock colony. This entailed that aphid nymphs
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41 116 in blocks 7-10 were on average 24 h older when attacked than nymphs in blocks 1-6. Any
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117 additional variation this might have caused entered the block factor of our analyses. Ten
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46 118 days after exposure to parasitoids, mummies were clearly visible and counted.
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53 121 All statistical analyses were carried out in R 2.7.1 (R Development Core Team 2008).
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122 The proportion of aphids exposed to wasps that were mummified served as our estimate
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58 123 of susceptibility to parasitoids and was analysed using a generalised linear model with
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60 124 logit link and – due to overdispersion – quasibinomial errors. We tested for the effects of
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125 block, infection with R. insecticola, clone and the infection × clone interaction. Adult
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6 126 body mass and daily fecundity were analysed with linear models testing for the same
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8 127 effects.
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13 129 3. RESULTS
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18 131 parasitoid A. colemani (Table 1). The originally resistant clone 5.15 became susceptible
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25 134 endosymbiont (Fig. 1). The significant difference among the four aphid clones is largely
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30 136 clones when uninfected with R. insecticola (Table 1, Fig. 1). The block effect was also
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32 137 significant, but there was no significant infection × clone interaction, showing that R.
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138 insecticola had a similar effect in different, even heterospecific genetic backgrounds.
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37 139 Infection with R. insecticola had a positive effect on aphid adult mass, which also
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differed significantly among the four aphid clones (Table 1, Fig. 2). However, these
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42 141 differences did not translate into variation in daily fecundity, which was similar for all
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44 142 clones and not affected by R. insecticola (Table 1, Fig. 2).
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51 145 We show that an isolate of the endosymbiotic bacterium R. insecticola from an Australian
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56 147 have been reported previously for two other aphid symbionts, H. defensa and – to a lesser
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150 This is fascinating, if not surprising, given that under faithful vertical transmission, the
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6 151 evolutionary interests of host and symbiont are well aligned. Thus, R. insecticola should
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8 152 be added to the list of endosymbionts capable of defending aphids against parasitoids,
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153 even if most strains of this bacterium may not possess this ability (Oliver et al. 2003;
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13 154 Vorburger et al. 2009). In H. defensa, variation in the level of defence that different
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15 155 strains provide has been linked to the copy number of the toxin-encoding bacteriophage
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18 156 APSE, which is required for the protective phenotype (Oliver et al. 2009). Whether the
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20 157 same is true for R. insecticola remains to be investigated. First PCR screens did not
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22 158 provide any evidence for APSE in the protective strain of R. insecticola described here
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25 159 (Nancy Moran, pers. comm.), but this does not exclude the possibility of other phage
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27 160 variants being involved.
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161 Considering the strong benefit provided by defensive endosymbionts, it is surprising
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32 162 that they are not more common in aphid populations. Possibly, there are also costs of
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34 163 harbouring such bacteria. Here we found no evidence for this assumption, as aphids were
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37 164 somewhat heavier and equally fecund when infected with R. insecticola. However, a
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39 165 study by Oliver et al. (2008) on H. defensa indicates that costs may only be expressed
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41 166 under more realistic conditions. In the case of R. insecticola, we only have evidence for
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167 induced costs of symbiont-conferred resistance, as individuals of the naturally infected
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46 168 clone 5.15 of M. persicae suffer from a strongly reduced fecundity after successfully
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48 169 resisting a parasitoid attack (Vorburger et al. 2008). This would at least reduce the
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51 170 benefits of harbouring R. insecticola.
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172 We thank D. Bopp for access to the microinjector, A. Gouskov for his help with aphid
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58 173 rearing, and N. Moran and two reviewers for comments on the manuscript. This work was
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60 174 supported by the Swiss National Science Foundation (grant 3100A0-109266 to CV).
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6 176
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8 177 Douglas, A. E. 1998 Nutritional interactions in insect-microbial symbioses: Aphids and
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178 their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43, 17-37. (doi:
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13 179 doi:10.1146/annurev.ento.43.1.17).
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15 180 Ferrari, J., Müller, C. B., Kraaijeveld, A. R. & Godfray, H. C. J. 2001 Clonal variation
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18 181 and covariation in aphid resistance to parasitoids and a pathogen. Evolution. 55,
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20 182 1805-1814. (doi: 10.1111/j.0014-3820.2001.tb00829.x).
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22 183 Ferrari, J., Darby, A. C., Daniell, T. J., Godfray, H. C. J. & Douglas, A. E. 2004 Linking
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25 184 the bacterial community in pea aphids with host-plant use and natural enemy
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186 Henter, H. J. & Via, S. 1995 The potential for coevolution in a host-parasitoid system. I.
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32 187 Genetic variation within an aphid population in susceptibility to a parasitic wasp.
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37 189 Moran, N. A., Russell, J. A., Koga, R. & Fukatsu, T. 2005 Evolutionary relationships of
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39 190 three new species of Enterobacteriaceae living as symbionts of aphids and other
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41 191 insects. Appl. Environ. Microbiol. 71, 3302-3310. (doi: 10.1128/AEM.71.6.3302-
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48 194 symbionts in aphids confer resistance to parasitic wasps. Proc. Natl. Acad. Sci. U. S.
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53 196 Oliver, K. M., Campos, J., Moran, N. A. & Hunter, M. S. 2008 Population dynamics of
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6 200 factors required for protection in a symbiotic mutualism. Science 325, 992-994. (doi:
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202 R Development Core Team. 2008 R: a language and environment for statistical
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13 203 computing. http://cran.r-project.org.
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15 204 Scarborough, C. L., Ferrari, J. & Godfray, H. C. J. 2005 Aphid protected from pathogen
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18 205 by endosymbiont. Science 310, 1781-1781. (doi: 10.1126/science.1120180).
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20 206 Schmidt, M. H., Lauer, A., Purtauf, T., Thies, C., Schaefer, M. & Tscharntke, T. 2003
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27 209 Tsuchida, T., Koga, R., Sakurai, M. & Fukatsu, T. 2006 Facultative bacterial
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210 endosymbionts of three aphid species, Aphis craccivora, Megoura crassicauda and
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34 212 Zool. 41, 129-137. (doi: 10.1303/aez.2006.129).
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37 213 von Burg, S., Ferrari, J., Müller, C. B. & Vorburger, C. 2008 Genetic variation and
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39 214 covariation of susceptibility to parasitoids in the aphid Myzus persicae – no evidence
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41 215 for trade-offs. Proc. R. Soc. Lond. B 275, 1089-1094. (doi: 10.1098/rspb.2008.0018).
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216 Vorburger, C., Gouskov, A. & von Burg, S. 2008 Genetic covariation between
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46 217 effectiveness and cost of defence in aphids. Biol. Lett. 4, 674-676. (doi:
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48 218 doi:10.1098/rsbl.2008.0382).
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51 219 Vorburger, C., Sandrock, C., Gouskov, A., Castañeda, L. E. & Ferrari, J. 2009 Genotypic
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53 220 variation and the role of defensive endosymbionts in an all-parthenogenetic host-
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221 parasitoid interaction. Evolution. 63, 1439-1450. (doi: 10.1111/j.1558-
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58 222 5646.2009.00660.x).
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224 Figure captions
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8 226 Fig. 1. Susceptibility of experimental lines of aphids to the parasitoid Aphidius colemani.
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227 Each bar represents the mean of 10 assays.
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15 229 Fig. 2. Adult mass (top) and daily fecundity (bottom) of four aphid clones in the presence
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18 230 and absence of the bacterial endosymbiont Regiella insecticola.
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Table 1. Generalised linear model results for the proportion of aphids mummified and linear model results for adult mass and daily fecundity.
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7 Proportion mummified Adult mass Daily fecundity
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10 Source of variation d.f. Deviance F P MS F P MS F P
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126.16 3.396 0.002 0.008 1.368 0.222 0.538 0.521 0.854
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Infection with R. insecticola 1 316.87 rR
76.776 < 0.001 0.038 6.435 0.014 0.002 0.002 0.969
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Aphid clone 3 40.08 3.237
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Infection × clone
Residual
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0.865 0.464 0.630
1.032
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Fig. 1. Susceptibility of experimental lines of aphids to the parasitoid Aphidius colemani. Each bar
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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)
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