1
Supplementary materials
2
Rapid evolution of Wolbachia incompatibility types
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Olivier Duron , Jennifer Bernard, Célestine M. Atyame, Emilie Dumas and Mylène Weill
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4
Abbreviations Culex pipiens lines wPip strains
Origins
References
Lv
LaVar
wPip(Lv)
France
(Duron et al. 2006a)
Is
Istanbul
wPip(Is)
Turkey
(Duron et al. 2006a)
Ko
Kol
wPip(Ko)
Crete
(Duron et al. 2006a)
Tn
Tunis
wPip(Tn)
Tunisia
(Duron et al. 2006a)
Sl-TC
SlabTC
uninfected
derived from the Slab line (California) (Duron et al. 2006a)
5
6
Table S1. Description of the Culex pipiens lines and the wPip strains.
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7
Genes
Culex pipiens (nuclear genome)
Putative products
Primers (5'-3')
Size (bp)
References
ace-2
Acetylcholinesterase 2 (AChE2)
700
(Bourguet et al. 1998)
Ester2
Carboxylester hydrolase
F1457-GAGGAGATGTGGAATCCCAA
B1246-TGGAGCCTCCTCTTCACGGC
Bdir1530-CTCCAGATCAACCCTTC
MMI_R-CAGCTTCGGGTCGATCATCAT
1100
(Ben Cheikh et al. 2008)
Cytochrome b
10366F-CTTTATTAGTAACTGTAAAAATTAC
11217R-ACTAAAGGATTAGCAGGAATGA
852
(Atyame et al. 2011a)
pk1
Ankyrin domain protein
1,334-1,349
(Duron et al. 2007; Sinkins et al. 2005)
ank2
Ankyrin domain protein
313-511
(Duron et al. 2007)
vrlC (=GP15)
Phage related probable secretory protein
1,511-1,538
(Atyame et al. 2011b; Duron et al. 2006b)
WPa_679
Guanylate kinase
F-CCACTACATTGCGCTATAGA
R-ACAGTAGAACTACACTCCTCCA
F-CTTCTTCTGTGAGTGTACGT
R2-TCCATATCGATCTACTGCGT
F1-ACCATTACAGAACTTGAGGA
R1-TAGACGTTCATAGGCAACCA
F2-ACCTGACTCTGCAGTACTTGA
R2-ACTGCTTCTCTCATAAATTCA
F- TATCCTCTCCTTCTGGAGCT
R- CTTCCATTGAGGGAGGTAGT
360
This study
16S rRNA
Small ribosomal subunit
ChF-TACTGTAAGAATAAGCACCGGC
ChR-GTGGATCACTTAACGCTTTCG
396
(Zchori-Fein & Perlman 2004)
yaeT
Outer membrane protein assembly factor
yaeTf-GCATACGGTTCAGACGGGTTTG
yaeTr-GCCGAAACGCCTTCAGAAAAG
473
(Duron et al. 2010)
16S rRNA
Small ribosomal subunit
SpixoF-TTAGGGGCTCAACCCCTAACC
SpixoR-TCTGGCATTGCCAACTCTC
810
(Duron et al. 2008)
gltA
Citrate synthetase
RICS741F-CATCCGGAGCTAATGGTTTTGC
RCIT1197R-CATTTCTTTCCATTGTGCCATC
ca.450
(Davis et al. 1998)
Culex pipiens (mitochondrial genome)
cytB
Wolbachia (wPip)
Cardinium
Arsenophonus
Spiroplasma (ixodetis group)
Rickettsia
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Table S2. Genes and primers used for detection diagnosis tests.
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3
11
Allelic profiles
Mosquito lines
Culex pipiens
n
nuclear genes
ace-2
Wolbachia (wPip)
mitochondrial gene
Ester2
cytb
pk1
ank2
vrlC
WPa_679
2005
Lv
10
a
a
pi7
c
e
b
a
Is
10
a
b
pi12
d
c
d
a
Ko
10
a
b
pi4
a
a
a
a
Tn
10
a
b
pi4
a
a
a
b
Sl-TC
10
b
c
pi11
_
_
_
_
Lv
12
a
a
pi7
c
e
b
a
Is
12
a
b
pi12
d
c
d
a
Ko
12
a
b
pi4
a
a
a
a
Tn
12
a
b
pi4
a
a
a
b
Sl-TC
12
b
c
pi11
_
_
_
_
SlwLv
10
b
c
pi7
c
e
b
a
SlwIs
10
b
c
pi12
d
c
d
a
wKo
10
b
c
pi4
a
a
a
a
SlwTn
10
b
c
pi4
a
a
a
b
2009 (original nuclear background)
2009 (Sl-TC nuclear background)
Sl
12
13
Table S3. Allelic profiles of Culex pipiens lines and wPip strains. The allelic profiles of mitochondrial and wPip genes were assessed by direct
14
sequencing and the profiles of nuclear genes by PCR/RFLP tests. Nomenclatures for mitochondrial and wPip alleles are those used by Atyame et
15
al. (2011a). Sl-TC is a Wolbachia-uninfected line. n: number of individuals typed; dash: absence of PCR product.
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17
References
18
Atyame, C., Delsuc, F., Pasteur, N., Weill, M. & Duron, O. 2011a Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito.
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20
21
22
23
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Molecular Biology and Evolution 28 , 2761-2772.
Atyame, C., Duron, O., Tortosa, P., Pasteur, N., Fort, P. & Weill, M. 2011b Multiple Wolbachia determinants control the evolution of
cytoplasmic incompatibilities in Culex pipiens mosquito populations. Molecular Ecology 20 , 286-298.
Ben Cheikh, R., Berticat, C., Berthomieu, A., Pasteur, N., Ben Cheikh, H. & Weill, M. 2008 Characterization of a novel high-activity esterase in
Tunisian Populations of the mosquito Culex pipiens . Journal of Economic Entomology 101 , 484-491.
Bourguet, D., Foncesca, D., Vourch, G., Dubois, M. P., Chandre, F., Severini, C. & Raymond, M. 1998 The acetylcholinesterase gene ace: a
25
diagnostic marker of the pipiens and quinquefasciatus forms of the Culex pipiens complex. J. Amer. Mosq. Control Assoc. 14 , 390-
26
396.
27
28
29
30
31
32
Davis, M. J., Ying, Z., Brunner, B. R., Pantoja, A. & Ferwerda, F. 1998 Rickettsial relative associated with Papaya bunchy top disease. Current
Microbiology 26 , 80–84.
Duron, O., Bernard, C., Unal, S., Berthomieu, A., Berticat, C. & Weill, M. 2006a Tracking factors modulating cytoplasmic incompatibilities in
the mosquito Culex pipiens . Molecular Ecology 15 , 3061-3071.
Duron, O., Bouchon, D., Boutin, S., Bellamy, L., Zhou, L., Engelstadter, J. & Hurst, G. D. 2008 The diversity of reproductive parasites among
arthropods: Wolbachia do not walk alone. BMC Biology 6 , 27.
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35
36
37
38
39
40
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Duron, O., Boureux, A., Echaubard, P., Berthomieu, A., Berticat, C., Fort, P. & Weill, M. 2007 Variability and expression of ankyrin domain
genes in Wolbachia variants infecting the mosquito Culex pipiens. J Bacteriol 189 , 4442-8.
Duron, O., Fort, P. & Weill, M. 2006b Hypervariable prophage WO sequences describe an unexpected high number of Wolbachia variants in the
mosquito Culex pipiens. Proc Biol Sci 273 , 495-502.
Duron, O., Wilkes, T. E. & Hurst, G. D. 2010 Interspecific transmission of a male-killing bacterium on an ecological timescale. Ecol Lett 13 ,
1139-48.
Sinkins, S. P., Walker, T., Lynd, A. R., Steven, A. R., Makepeace, B. L., Godfray, H. C. & Parkhill, J. 2005 Wolbachia variability and host
effects on crossing type in Culex mosquitoes. Nature 436 , 257-60.
Zchori-Fein, E. & Perlman, S. J. 2004 Distribution of the bacterial symbiont Cardinium in arthropods. Mol Ecol 13 , 2009-16.
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Figure legends
43
Figure S1. Hatching rates from crosses (A) between ♀Lv and ♂Tn in 2005 and 2009, and
44
between ♀SlwLv and ♂SlwTn; and (B) between ♀Is and ♂Ko in 2005 and 2009, and between
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♀SlwIs and ♂SlwKo. y axis indicates hatching rates. The number of clutches observed (n) is
46
indicated for each cross (2000-3600 eggs were examined per cross). a and b represent
47
statistical groups (Wilcoxon two sided-tests).
48
49
Figure S2. Crossing relationships of a Wolbachia-uninfected line (Sl-TC) with four infected
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lines (Lv, Is, Ko and Tn) of Cx. pipiens in 2005 and 2009. A, crosses of uninfected males with
51
infected and uninfected females; B, crosses of uninfected females with infected males.
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Histograms give the distribution of hatching rates (HR) (x axis, HR; y axis, proportion of
53
clutches). The number of clutches observed (n) is indicated for each cross (a minimum of
54
1000 eggs was examined per cross). For each cross, no significant variation of HR was found
55
between 2005 and 2009 (Fisher exact tests; a refers to statistical group).
56
57
Figure S3. Hatching rates from crosses of (A) ♀Lv-sub12 and ♀Lv-sub10 with ♂Tn; and (B)
58
♀Is-sub5 and ♀Is-sub2 with ♂Ko. The number of clutches observed (n) is indicated for each
59
cross (1500-4200 eggs were examined per cross). y axis indicates hatching rates. a and b
60
represent statistical groups (Wilcoxon two sided-tests).
61
62
Figure S4. Hatching rates from reciprocal crosses between (A) Lv-sub12 and Lv-sub10, and
63
(B) Is-sub5 and Is-sub2. The number of clutches observed (n) is indicated for each cross (800-
64
3800 eggs were examined per cross). y axis indicates hatching rates. a represents statistical
65
group (Wilcoxon two sided-tests).
66
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67
Figure S5. Transgenerational survey of crosses (A) between ♀Lv-sub12 and ♂Tn males; (B)
68
between ♀Lv-sub10 and ♂Tn; (C) between ♀Is-sub5 and ♂Ko; and (D) between ♀Is-sub1
69
and ♂Ko. For each cross, no significant variation of hatching rates was observed over 10
70
generations but note in (C) the appearance of few compatible clutches (visualized by outliers).
71
The number of clutches observed (n) is indicated for each cross (1900-5400 eggs were
72
examined per cross). y axis indicates hatching rates. a represents statistical group for
73
transgenerational comparisons (Wilcoxon two sided-tests).
74
8
75
76
77
Figure S1
9
♀Sl-TC
80
2009
(n = 16)
2005
(n = 20)
2009
(n = 9)
2005
(n = 49)
2009
(n = 11)
2005
(n = 22)
2009
(n = 23)
a
a
a
a
a
a
a
a
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
1.0
0.8
0.6
0.4
0.2
0.0
2005
(n = 50)
0.00
0.25
0.50
0.75
1.00
78
2005
(n = 37)
2009
(n = 10)
2005
(n = 29)
2009
(n = 14)
2005
(n = 10)
2009
(n = 10)
2005
(n = 27)
2009
(n = 19)
2005
(n = 35)
2009
(n = 12)
a
a
a
a
a
a
a
a
a
a
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
1.0
0.8
0.6
0.4
0.2
0.0
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
♂Sl-TC
0.00
0.25
0.50
0.75
1.00
A
♀Lv
♀Is
♂Lv
♀Ko
♂Is
♀Tn
♂Ko
♀Sl-TC
B
♂Tn
79
Figure S2
10
81
A A AA
Lv[w
Pip(Lv)]
sublines
♀ Lv[w
sublines
♀♀Lv[w
Pip(Lv)]
sublines
♀Pip(Lv)]
Lv[w
Pip(Lv)]
sublines
Lv-sub12
Lv-sub10
♀ Lv-sub12
♀ Lv-sub10
♀♀Lv-sub12
♀♀Lv-sub10
♀ Lv-sub12
♀ Lv-sub10
n
=
21
14
n = 21n = 21
n = 21n = 14n = 14
nn==14
1.0
1.0
1.0
1.0
0.8
0.8
0.8
Tn[wPip(Tn)]
Pip(Tn)]
0.6
♂ Tn[w
0.6
♂♂Tn[w
♂ Pip(Tn)]
Tn[w
Pip(Tn)]
0.6
0.6
0.8
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.2
0.0
0.0
0.0
0.0
A BBB
a
b
a
b
bb
aa
♀ Is[w
sublines
♀Pip(Is)]
Is[w
Pip(Is)]
sublines
♀ Lv[w Pip(Lv)]
sublines
♀ Is[w
Pip(Is)]
sublines
1.0
0.8
♀ Is-sub5
♀ Is-sub1
♀♀
Is-sub5
♀ Is-sub1
♀ Lv-sub12
Lv-sub10
♀ Is-sub5
♀ Is-sub1
=n30
= 24
n = 21 n =n30
= 14 n =n24
=n30
=n24
b b b
1.0 1.0 1.0
b
0.8 0.8 0.8
0.6 0.6 0.6
Ko[w
Pip(Ko)]
0.6
♂ Ko[w
Pip(Ko)]
♂ Tn[w ♂
Pip(Tn)]
♂ Ko[w
Pip(Ko)]
0.4
0.4 0.4 0.4
0.2
0.2 0.2 0.2
0.0
0.0 0.0 0.0
a
a a a
82
83
84
Figure S3
11
A
♀ Lv-sub12
1.0
♀ Lv-sub10
n = 32
n = 16
a
a
B
1.0
0.8
♂ Lv-sub12
♂ Is-sub5
n = 20
n=7
a
a
n = 12
n = 31
0.6
0.4
0.4
0.2
0.2
0.0
0.0
n = 13
a
n = 26
a
1.0
0.8
85
♀ Is-sub1
0.8
0.6
1.0
♂ Lv-sub10
♀ Is-sub5
a
a
0.8
♂ Is-sub1
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
86
87
Figure S4
12
AA
♀ Lv-sub12
♀ Lv-sub12
1.0
0.8
♂ Tn[w
Pip(Tn)]0.6
♂ Tn[w
Pip(Tn)]
0.4
0.2
0.0
1.0
G1 G1
n = 21
n = 21
G2 G2
n = 19
n = 19
G3 G3
n = 22
n = 22
G4 G4
n = 30
n = 30
G5 G5
n = 18
n = 18
0.8
0.6
0.4
0.2
a
a
a
a
a
a
a
a
a
a
a
♀ Lv-sub12
♀ Lv-sub10
G1 G1
n = 14n = 21
1.0
1.0
0.8
0.8
♂ Pip(Tn)]
Tn[w Pip(Tn)]0.6
♂ Tn[w
0.6
0.4
0.4
0.2
0.2
0.0
0.0
a
G2 G2
n = 20n = 19
a
a
a
CC
G3 G3
n = 26n = 22
a
G4 G4
n = 21n = 30
a
a
G5 G5
n = 24n = 18
a
G10 G10
n = 26n = 36
a
a
a
a
♀ Is-sub5
♀ Is-sub5
G1 G1
n = 21n = 21
1.0
1.0
0.8
0.8
0.6
♂ Ko[w
Pip(Ko)]
♂ Ko[w
Pip(Ko)]
0.6
0.4
0.4
0.2
0.2
0.0
0.0
a
a
G2 G2
n = 30n = 30
a
G3 G3
n = 37n = 37
a
a
G4 G4
n = 51n = 51
a
G5 G5
n = 31n = 31
G10 G10
n = 48n = 48
a
a
a
a
a
a
DC
♀ Is-sub1
♀ Is-sub5
1.0
1.0
0.8
0.8
0.6
♂ Ko[w
♂ Pip(Ko)]
Ko[w Pip(Ko)]
0.6
0.4
0.4
0.2
0.2
0.0
0.0
G1 G1
n = 30n = 21
a
G2 G2
n = 24n = 30
a
G3 G3
n = 31n = 37
a
a
G4 G4
n = 27n = 51
a
G5 G5
n = 17n = 31
a
a
89
a
0.0
BA
88
G10 G10
n = 36
n = 36
G10 G10
n = 26n = 48
a
a
a
a
a
Figure S5
13