Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2010 A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids Vorburger, C ; Gehrer, L ; Rodriguez, P 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 peach-potato 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. 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 1 http://mc.manuscriptcentral.com/bl Submitted to Biology Letters Page 2 of 13 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 2 http://mc.manuscriptcentral.com/bl Page 3 of 13 Submitted to Biology Letters 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 3 http://mc.manuscriptcentral.com/bl Submitted to Biology Letters Page 4 of 13 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 .