Identifying potential drivers of haplotype frequency increase
The genomic region into which the ZZB-TE inserted does not show histone
signals of regulatory activation (H3K27ac, H3K9ac) or repression
(H3K9me3), and ATAC-seq data suggests it is not in an open chromatin
region [35]. However we took two in silico approaches to determine
whether the ZZB-TE insertion (748bp) carried putative regulatory
variants. The inserted region had 98.5% sequence identity to a putative
enhancer (2R:45966598-45966822) identified by homology withDrosophila melanogaster [35]. However, despite the
similarity, the insertion lacked enhancer-like combination of chromatin
marks identified in [35] and its potential regulatory role in nearby
genes is unclear. The second approach involved screening the ZZB-TE
inserted sequence for putative enhancers using iEnhancer-2L[36] and
iEnhancer-EL [37]. In a windowed analysis of 200bp with a 1bp step
across the entire length of ZZB-TE both predicted that some of the
windows would have strong enhancer activity, however the windows were
not-concordant, precluding further analysis.
Given that cytochrome P450 mediated resistance is commonly associated
with differential gene expression we performed transcription studies
within the Cyp6aa/Cyp6p cluster between the most contrasting
haplotypes, wild-type and triple mutant, present in the BusiaUg colony.
The group homozygous for the triple mutant haplotype significantly
overexpressed both Cyp6aa1 (2.23-fold, 95% CI: 1.73-2.90,
P=0.0003) and Cyp6p4 (2.57-fold, 95% CI 1.25-5.93, P=0.039)
compared to wild-type individuals. The ratio of expression ofCyp6aa 1 broadly reflected the expected pattern based on genotype
(ie 2:1.5:1 for triple mutant homozygotes: heterozygotes: wild type
genotypes, respectively. Figure 4). As a control we examined a
neighbouring, very commonly resistance-associated gene, Cyp6p3 ,
but triple mutant and wild type homozygotes did not differ significantly
in expression (1.33 fold, 95% CI 0.64-2.74, P>0.05).
To investigate whether resistance may be driven at least in part by an
effect of the allelic variant on metabolic activity of CYP6P4, we
expressed the wild-type (236I) and mutant (236M) forms in an E.
coli based recombinant protein system (Supplementary materials
Appendices 2 and 4). Both alleles were shown to be capable of
metabolizing class I (permethrin) and II (deltamethrin) pyrethroids but
there was no evidence that the mutant (236M) or wildtype (236I) alleles
had different rates of pyrethroid depletion. We also expressed the
duplicated P450 CYP6AA1, in an Sf9 -baculovirus protein expression
system. Again, metabolism assays demonstrated that the enzyme was
capable of metabolizing both deltamethrin and permethrin (Supplementary
materials Appendices 3 and 4). Depletion of deltamethrin was 36.6%
greater (SE= 3.79) in the presence of NADPH than in the control (t-test:
t=-9.67; d.f. = 8; P= 9.6 x10-6), demonstrating that
CYP6AA1/CPR is capable of metabolizing deltamethrin in vitro .
Similarly, permethrin was metabolised by CYP6AA1/CPR, with permethrin
being depleted by 22.4% (SE = 0.63) compared to the control without
NADPH (t-test: t= -31.08; d.f. = 14; P= 2.55 x10-14).
Given clear evidence of increased expression of both Cyp6aa1 andCyp6p4 in the triple mutant haplotype and the ability of both
enzymes to metabolise pyrethroids in vitro , we investigated
whether the mutations were significantly associated with resistancein vivo. Exposure of An. gambiae females from Busia,
Uganda and Nord Ubangi, DRC to new LLINs in cone assays resulted in
negligible mortality to the pyrethroid only LLINs, Olyset and Permanent
2.0 (Figure 5). Simultaneous exposure to pyrethroid plus the P450
inhibitor PBO in Olyset + and the top of Permanent 3.0 nets resulted in
a marked reduction in resistance, demonstrating that the resistance
phenotype is substantially mediated by P450s. We performed laboratory
backcrosses between additional mosquitoes from Busia with the pyrethroid
susceptible Mbita colony, and found that the triple mutant haplotype was
significantly associated with resistance to the most commonly used type
II pyrethroids in LLINs: deltamethrin (Fisher’s exact test
p=3.2x10-6) and alphacypermethrin (Fisher’s exact test
p=5.9x10-7) resistance although not to permethrin
(Fisher’s exact test p=0.06) nor, as a control, DDT (Fisher’s exact test
p=0.84) (Table 1) in WHO tube assays. Similarly, specimens collected in
2016 from the DRC showed a strong association between the triple mutant
genotype and survival rate 24 hours post-exposure to either 0.05%
deltamethrin for 1 hour or 3-minute exposures to deltamethrin-treated
sides of a new PermaNet 3.0 net (Table 2). No association was found in
samples exposed to permethrin (24 hour WHO tube assay) or
permethrin-treated Olyset Plus nets (3-minute WHO cone assay) (Table 2).
Complete linkage of the three mutants in the BusiaUG colony and the DRC
wild caught collections precludes determination of the relative
contribution of each of the three mutations to the resistance phenotype
but taken together these results demonstrate a strong impact of the
triple mutant on the efficacy of pyrethroid resistance.