Discussion
We have identified a sequential series of fitness-augmenting mutations
in An. gambiae culminating in a triple-mutant haplotype with a
large effect on pyrethroid resistance and which is spreading rapidly
across East and Central Africa. The mutation that is probably the oldest
in this series, the insertion of ZZB-TE, was first detected in 2004 in
the malaria-endemic area around Lake Victoria, with theCyp6p4-236M SNP evident in 2005 samples and the third, a
duplication in Cyp6aa1 detected in 2008. In samples collected
only five years later, the triple mutant was detected hundreds of
kilometres away in the DRC. These patterns suggest both a large fitness
advantage arising from the triple mutant, and a frightening speed at
which resistance-conferring mutations are able to spread within and
across populations.
Second generation nets treated with the synergist PBO were shown to be
much more effective than conventional nets in killing mosquitoes in
populations where the triple mutant haplotype is present (Figure 5).
Therefore the use of PBO bednets should be prioritised in the regions
where this mutation is present. A strong corollary of this finding comes
from the cluster randomised control trial conducted in Uganda, where the
mutation is at high frequency, which demonstrated that malaria parasite
prevalence in children <10 years old (12% vs 14%; Prevalence
ratio = 0·84, 95% CI 0·72–0·98; P=0·029) and mean number of mosquitoes
(Density ratio=0·25, 95% CI 0·18–0·3; P <0·0001) per house
were significantly lower in villages that had received PBO LLINs
relative to standard LLINs [4]. There is some evidence that the
haplotype may be less strongly associated with resistance to permethrin
than deltamethrin (and perhaps also alphacypermethrin), although both
pyrethroids were metabolised by Cyp6aa1 and Cyp6p4 .
Our results highlight the importance of gene duplications for the
evolution of insecticide resistance. In An. gambiae , duplications
have recently been shown to be concentrated in regions associated with
metabolic resistance, and over 40 such duplications have been described
across the genome
[20]. Thirteen different duplications have so far been described
that encompass Cyp6aa1 (Figure 1), both in West and East Africa
and in the two sister-species An. gambiae ss and An.
coluzzii [20]. It seems likely that these other Cyp6aa1duplications are also associated with pyrethroid resistance. For
example, in An. coluzzii sampled from a highly
insecticide-resistant population from Cote d’Ivoire, greater than 95%
of individuals had Cyp6aap duplications (Supplementary materials
Appendix 5). Five different duplications were observed in the
collections from 2012 and 2017 and, whilst we detected no association
with pyrethroid resistance, two duplications (Cyp6aap-Dup7 andCyp6aap-Dup14 ) showed significant increases in frequency over
time. Moreover the total number of CNVs per sample (measured as presence
of each of the 5 duplications, summed for each sample) increased
significantly from an average of 1.59/ individual in 2012 to 2.08 in
2017 (Mann Whitney U test; P<0.0001); the mean number of
duplications >2 indicates that there are multiple CNVs on
the same haplotype. Duplications of the Cyp6aa1 orthologue have
been found in another important malaria vector, An. funestus ,
from west and central Africa [21]. The Cyp6aa1 orthologue inAn. funestus, which shares an 87% identity with An.
gambiae, was also observed to metabolize permethrin and deltamethrin
and, when expressed in transformed Drosophila , was associated
with significant increases in resistance to both permethrin and
deltamethrin relative to control mosquitoes [21]. This evolution of
multiple Cyp6aa1 duplications suggests this is an important
Africa wide resistance mechanism
There are now several cases of insecticide resistance evolution where an
initial mutation in a genomic region is followed by the spread of
additional mutations on the resistant haplotype background [14,
38-42]. It is not yet clear whether the sequence of mutations that we
have identified rely on each-other for effect, and thus could only have
spread sequentially, or whether each additional mutation coincidentally
appeared on the background of an already common mutant haplotype. In the
case of Cyp6p4-236M and Cyp6aap-Dup1 it seems that the
latter is more likely, although we cannot exclude the possibility that
the duplication affects the regulation of Cyp6p4 . In contrast,
the putative enhancer inserted with the ZZB transposon may affect either
or both of Cyp6p4 and Cyp6aa1 , and may thus interact with
the other two mutations in ways that have yet to be determined.
Transposable elements can sometimes affect gene expression of
neighbouring genes [38] and are abundant in mosquito genomes [43,
44]. Interestingly, in the common house mosquito, Culex
pipiens , TEs have been found in the flanking regions of theEster locus, a genomic region in which many independent gene
duplications have arisen and spread worldwide in response to selection
from organophosphates [45]. Clearly, both TEs and gene duplications
are an understudied, yet common source of variation that may have
important implications for vector control efforts. The appearance and
rapid spread of the three mutations described here is broadly coincident
with the scale up of LLIN coverage in DRC, Kenya and Uganda. The
haplotype is a strongly predictive marker of high-level resistance to
pyrethroids, is easily screened with a single diagnostic assay and we
suggest should be used for both insecticide resistance monitoring
strategies and for informing LLIN selection.[6, 29, 46].