Supporting Information
Table S1. PCR primers used in this project.
Target gene
Primer pair (5’ to 3’)
(Fragment
Annealing
Target group
(ºC)
size)
10F: AGTTTGATCATGGCTCAGATTGa
16S rRNA
Most bacteria
1507R: TACCTTGTTACGACTTCACCCCAGa
(~1500 bp)
27F: GAGAGTTTGATCCTGGCTCAGb
16S rRNA
1492R: GGTTACCTTGTTACGACTTb
(~1470 bp)
Most bacteria
559F: CGTGCCAGCAGCCGCGGTAATACc
temp
16S-ITS-35R
Most bacteria
(>1000 bp)
(not Wolbachia)
60
55
58
35R: CCTTCATCGCCTCTGACTGCd
p58IV_F: AAAGGTTTACATTCACCAAGTCGe
P58
Spiroplasma
p58IV_R: ATTGTTCATTAACTTTATCTTGTGGe
53
(362 bp)
wspF:
TGGTCCAATAAGTGATGAAGAAACTAGCTAf
wsp
Touchdown
Wolbachia
wspR:
(~600 bp)
65–55
AAAATTAAACGCTACTCCAGCTTCTGCACf
HCO2198: TAAACTTCAGGGTGACCAAAAAAT g
COI
Most
LCO1490: GGTCAACAAATCATAAAGATATTGg
(~709 bp)
invertebrates
45
a Munson et al. (1991)
b Lane (1991)
c Russell et al. (2003)
d Mateos et al. (2006)
e Xie et al. (2010)
f Jeyaprakash & Hoy (2000)
g Folmer et al. (1994)
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Table S1 Number of flies per isoline, Spiroplasma-infection state, and sex used to stock the initial generation of each
bottle (replicate).
Isoline ID
Spiroplasma infection
state
Sex
No. individuals in S+
population
No. individuals in
S-population
…
1
S–
S+
5
S–
S+
Total
S–
S+
F M
F
M F M
F
M F M
F
M
5
5
5
5
5
5
5
100
10 10
100
5
5
5
10 10
10 10
2
5
5
Figure S1. Experimental Setting. Columns indicate fly vials and circles represent population
bottles. S = Spiroplasma infection treatment; Lh = Leptopilina heterotoma treatment.
3
4
Figure S2. Vertical transmission efficiency of Spiroplasma in females from the S+Lh+ and S+Lhtreatments in all generations. S = Spiroplasma, Lh = Leptopilina heterotoma treatment. Error bar:
standard error. Note: G4 is lacking data because the selected replicate used for this generation had
lost Spiroplasma infection.
References
Folmer O, Black M, Hoeh W, Lutz R & Vrijenhoek R (1994) DNA primers for amplification of
mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol
Biotechnol 3: 294-299.
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Jeyaprakash A & Hoy MA (2000) Long PCR improves Wolbachia DNA amplification: wsp
sequences found in 76% of sixty-three arthropod species. Insect Mol Biol 9: 393–405.
Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial
systematics,(Stackebrandt E & Goodfellow M, eds.), pp. 115-175. John Wiley and Sons, New
York, NY.
Mateos M, Castrezana SJ, Nankivell BJ, Estes AM, Markow TA & Moran NA (2006) Heritable
endosymbionts of Drosophila. Genetics 174: 363-376.
Munson MA, Baumann P, Clark MA, Baumann L, Moran NA, Voegtlin DJ & Campbell BC
(1991) Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four
aphid families. J Bacteriol 173: 6321-6324.
Russell JA, Latorre A, Sabater-Munoz B, Moya A & Moran NA (2003) Side-stepping secondary
symbionts: widespread horizontal transfer across and beyond the Aphidoidea. Mol Ecol 12:
1061-1075.
Xie J, Vilchez I & Mateos M (2010) Spiroplasma bacteria enhance survival of Drosophila hydei
attacked by the parasitic wasp Leptopilina heterotoma. PLoS ONE 5: e12149.
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