Abstract
BackgroundThe recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined.MethodsThe APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis.ResultsAPL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus.ConclusionsAPL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.
Highlights
The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs
Anopheles stephensi samples used for APL1 population variation analysis were 6 individuals from a colony initiated at Chabahar, Iran in 2011, 6 individuals from a colony initiated at Bandar-Abbas, Iran in 2008 and 1 wild-caught individual from Bandar-Abbas
Phylogeny of APL1 gene expansion from a unique ancestor Recent in silico annotation of the An. stephensi reference genome detected a single APL1 gene [19]. This is in contrast to the Gambiae species complex, where APL1 is comprised of a family of three paralogs, APL1A, APL1B and APL1C, with distinct roles in immunity [14, 16]
Summary
The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. An important Asian DVS is Anopheles stephensi, which has recently been recognized as an invasive vector species, expanding disease transmission along with its geographical range [2, 3]. Among human-feeding DVS, there is apparent variation in vectorial capacity, suggested by large geographical differences in human malaria infection prevalence, with about 90% of global prevalence located in Africa [6]. Some of this global geographical variation could be caused by ecology, if some niches, for example in humid sub-Saharan Africa, are favorable to mosquito abundance and longevity, promoting malaria transmission [7,8,9]. Several tens of Anopheles genes are known from laboratory studies to control malaria infection of the vector, but involvement of these genes in modulating natural transmission has not been confirmed by genetic association in the natural vector population
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