Forward primer Reverse primer Expected size (bp ...

Table S1: List of primers used in the study

Forward primer qRT-PCR primers GTTTGAAGCAGTTGCCATACTACGAGG GSTe2 CYP6P9a

CAGCGCGTACACCAGATTGTGTAA

CYP6P9b

CAGCGCGTACACCAGATTGTGTAA

Reverse primer TCAAGCTTTAGCATTTTCCTCCTTTTTGG C TCA CAA TTT TTC CAC CTT CAA GTA ATT ACC CGC TTA CAC CTT TTC TAC CTT CAA GTA ATT ACC CGC GTAGATGGTGCCAAGGATGG ACCGGATGCATACAGTGACA TCCGAGTTCATTTCCAGCTC

Expected size (bp) 101 92 97

CAAATTTAAAGAGTGCGCTAGG 115 CYP9J11 TTAAACCCAAAAGCCAATCG 111 Actin GTGTTCGGTTCCAAGGTGAT 98 RSP7 Primers for functional analysis AGATCTATGACCAAGCTAGTTCTGTAC TCTAGATCAAGCTTTAGCATTTTCCTCCT GSTe2 ACGCT TTTTGGC GAATTCCATATGACCAAGCTAGTTCTG CCGCTCGAGTCAAGCTTTAGCATTTTCC TACACGCT TCCTTTTTGGC CYP9J11 ATG GAG ATC GAT TTG ATG GTT CT TTA CAT ACT AAC TTC GTT ATC TTT CTT TCT CYP9J11Full AGG GGAATTCCATATGAAAAAGACAGCTA ompA+2F TCGCG ompA+2CYP9J11_F AACCATCAAATCGATCTCCATCGGAGC GGCCTGCGCTACGGTAGCGAA TCTAGATCTAGATTACATACTAACTTCGTTATCTT ompA+2CYP9J11_R TC Primers used for amplification for polymorphism analysis ATCCCTAACTATTAAAAGGCAAT TCACAATTTTTCCACCTTCAAGTAATTACCCGC CYP6P9a CATACTCATAATAACTAGACGCG TTACACCTTTTCTACCTTCAAGTAATTACCCGC CYP6P9b In red is restriction site for BglII; in green is for XbaI; blue is for NdeI; purple is for XhoI; EcoRI is dark brown.

2.2 1.8

Table S2: Detoxification genes commonly up-regulated only in two countries between Uganda (UG), Malawi (MAL) and Benin (BN) Probe Name CUST_13921_PI426302897 CUST_11037_PI426302897 CUST_12461_PI426302897 CUST_6368_PI426302897 CUST_15244_PI426302897 CUST_13481_PI426302897 CUST_8354_PI426302897 CUST_3731_PI406199772 CUST_1088_PI406199772 CUST_15331_PI426302897 CUST_3736_PI406199772 CUST_12666_PI426302897 CUST_7722_PI426302897

Afun013921 Afun011037 Afun012461 Afun006368 Afun015244 Afun013481 (GSTe1) Afun008354 (GSTD3) CD577517.1 EE590086.1 Afun015331 CD577515.1 Afun012666 Afun007722

CUST_5005_PI406199798 CUST_574_PI406199772 CUST_4392_PI426302897 CUST_143_PI426302897 CUST_13218_PI426302897 CUST_10836_PI426302897 CUST_45_PI426302897 CUST_13870_PI426302897 CUST_12777_PI426302897 CUST_9_PI426302915 CUST_9492_PI426302897

AGAP003680-RA EE589639.1 Afun004392 Afun000143 Afun013218 Afun010836 Afun000045 (GSTe2) Afun013870 Afun012777 CYP6M4.seq Afun009492

CUST_15523_PI426302897

Afun015523

AGAP006709-PA AGAP003581-PA AGAP000288-PA AGAP003785-PB AGAP000820-PA AGAP009195-PA AGAP004382-PA

AGAP001039-PB AGAP002429-PA AGAP009850-PA AGAP003680RA

UG 49.5 8.0 6.8 6.4 5.9 5.6 5.1 4.7 4.5 3.9 3.8 3.7 3.6

MAL

BN 27.4 13.1 10.5 3.2 4.1 2.3 4.3 2.1 2.6 29.4 3.3 3.8 2.8

Description chymotrypsin 1 alcohol dehydrogenase alcohol dehydrogenase glucose dehydrogenase cuticular protein rr-1 family glutathione-s-transferase gst glutathione transferase cuticle protein d7-related 1 protein cytochrome p450 307a1 cuticle protein cytochrome p450 abc transporter

3.4 5.4 2.6 2.7 2.3 3.7 12.2

AGAP001722-PA

3.5 3.2 2.7 2.4 2.3 2.3 2.1 14.8 10.3 9.2 8.8

7.5 2.1 5.3 5.9

abc transporter d7-related 1 protein cytochrome p450 cytochrome p450 cyp9k1 cytochrome p450 esterase b1 glutathione-s-transferase gst sulfotransferase cytochrome p450 cytochrome p450 carboxylesterase

AGAP010581-PA

5.7

3.1

abc transporter

AGAP008213-PA AGAP000818-PA AGAP000284-PA AGAP006228-PA AGAP009194-PA AGAP012697-PA AGAP009246-PA

CUST_979_PI426302897 CUST_1149_PI406199772 CUST_3836_PI406199772 CUST_13272_PI406199769 CUST_3830_PI406199772 CUST_11963_PI426302897 CUST_4920_PI406199772 CUST_1150_PI406199772 CUST_13273_PI406199769 CUST_7_PI426302915 CUST_1687_PI406199772 CUST_7127_PI426302897 CUST_7008_PI426302897 CUST_5935_PI406199769 CUST_7482_PI426302897 CUST_12478_PI426302897 CUST_7696_PI406199798 CUST_3394_PI426302897 CUST_7499_PI426302897

Afun000979 EE590022.1 CD577459.1 combined_c6791 CD577462.1 Afun011963 BU038983.1 EE590022.1 combined_c6791 CYP6M3.seq EE589439.1 Afun007127 Afun007008 combined_c3002 Afun007482 Afun012478 AGAP008141RA___3R Afun003394 Afun007499 (GSTD1-5)

CUST_27_PI406199775 CUST_30_PI406199775 CUST_25_PI406199775 CUST_1096_PI406199769 CUST_26_PI406199775 CUST_13614_PI426302897 CUST_13328_PI426302897 CUST_8241_PI406199769

CYP6P9a CYP6P9b CYP6P9a combined_c557 CYP6P9a Afun013614 Afun013328 combined_c4173

AGAP006711-PA

AGAP006220-PA

AGAP009241-PA AGAP012156-PA AGAP002693-PA AGAP005757-PA AGAP008141RA AGAP000284-PA AGAP004164-PA

AGAP003446-PA AGAP000384-PA

4.5 4.4 4.3 4.2 4.2 4.0 3.8 3.7 3.6 2.9 2.9 2.6 2.6 2.4 2.4 2.2

3.7 2.4 3.6 4.8 2.7 2.8 2.6 2.3 4.2 10.1 5.7 2.4 2.2 2.7 2.3 3.1

chymotrypsin 1 d7-related 1 protein cuticle protein cytochrome p450 cuticle protein aldehyde oxidase cuticle protein d7-related 1 protein cytochrome p450 cytochrome p450 d7-related 1 protein cytochrome p450 ABC transporter superoxide dismutase ABC transporter carboxylesterase

2.2 2.1 2.1

8.5 2.4 2.9

argininosuccinate lyase cytochrome p450 glutathione transferase

39.4 24.0 16.3 11.8 9.8 8.3 8.0 4.8

4.3 4.1 3.4 3.8 2.7 4.0 2.2 3.7

cytochrome p450 cytochrome p450 cytochrome p450 trypsin cytochrome p450 zinc finger protein 616 zinc finger protein 650 glycoprotein 93

CUST_200_PI426302897 CUST_1823_PI406199769 CUST_431_PI406199788 CUST_10614_PI426302897 CUST_1182_PI406199772 CUST_7646_PI426302897

Afun000200 combined_c920 gb-CYP6M3 Afun010614 EE589982.1 Afun007646

AGAP004038-PA

AGAP006380-PA AGAP006225-PA

3.5 3.4 2.9 2.5 2.3 2.0

2.2 9.4 2.6 3.0 2.4 4.0

chorion peroxidase glutathione-s-transferase cytochrome p450 ABC transporter d7-related 1 protein aldehyde oxidase

Table S3: Summary statistics for polymorphism of CYP6P9a and CYP6P9b between the FANG susceptible and the resistant strains from Malawi and Mozambique Samples N S h CYP6P9A Uganda Alive 14 41 7

Syn

Nonsyn

 (k)

D (Tajima)

26

8

1.07ns

1.4*

Uganda dead All Uganda

8 30

6 14

-0.14ns -0.35ns

-0.44ns 0.88ns

13 5 61 20

4 33

2 16

0.008 (16.05) 0.0024 (4.8) 0.0061 (12.1) 0.001(1.98) 0.01(20.1)

-1.65ns 1.4 ns

-1.98 ns 0.05 ns

42 2 43 3

22 23

7 7

-2.45*** -2.34***

-3.5** -3.1**

30 45 4 20 12 2 50 73 6

25 6 44

7 3 12

0.003(5.25) 0.0029 (6.26) 0.003 (5.5) 0.0007(1.2) 0.012(21.43 )

12 15 8 26 50 15

Malawi 20 Total 46 CYP6P9b Uganda Alive 16 Uganda dead 14 All Uganda Malawi Total

-1.89* -2.3** 0.98ns

D* (Fu and Li)

1.19ns -3.5** 1.09ns

N= number of sequences (n); S, number of polymorphic sites; Syn, Synonymous mutations; Nonsyn, Nonsynonymous mutations; , nucleotide diversity (k= mean number of nucleotide differences); Tajima’s D and Fu and Li’s D statistics, ns, not significant.