Polymorphism at the defensin gene in the Anopheles gambiae complex: Testing different selection hypotheses

Abstract

Genetic variation in defensin, a gene encoding a major effector molecule of insects immune response was analyzed within and between populations of three members of the Anopheles gambiae complex. The species selected included the two anthropophilic species, An. gambiae and An. arabiensis and the most zoophilic species of the complex, An. quadriannulatus. The first species was represented by four populations spanning its extreme genetic and geographical ranges, whereas each of the other two species was represented by a single population. We found (i) reduced overall polymorphism in the mature peptide region and in the total coding region, together with specific reductions in rare and moderately frequent mutations (sites) in the coding region compared with non-coding regions, (ii) markedly reduced rate of non-synonymous diversity compared with synonymous variation in the mature peptide and virtually identical mature peptide across the three species, and (iii) increased divergence between species in the mature peptide together with reduced differentiation between populations of An. gambiae in the same DNA region. These patterns suggest a strong purifying selection on the mature peptide and probably the whole coding region. Because An. quadriannulatus is not exposed to human pathogens, identical mature peptide and similar pattern of polymorphism across species implies that human pathogens played no role as selective agents on this peptide.

Introduction

The completion of the sequencing of the genome of Anopheles gambiae (Holt et al., 2002), together with the successful germ line transformation of this mosquito (Grossman et al., 2001), and the identification of key molecules and genes affecting susceptibility of mosquitoes to Plasmodium under laboratory conditions (Barillas-Mury et al., 1996, Blandin et al., 2004, Osta et al., 2004a, Osta et al., 2004b) provide strong support for malaria control via the introduction and spread of refractory genes into vector populations (Collins et al., 2000). Immune-response molecules are particularly promising as determinants of vector susceptibility and have been the focus of a number of recent studies (Christophides et al., 2002, Dimopoulos, 2003, Osta et al., 2004a, Osta et al., 2004b, Meister et al., 2005). Some evidence suggests that Anopheles susceptibility to Plasmodium depends on the specific genotype of the vector and the parasite (Tahar et al., 2002, Lambrechts et al., 2005). Such finely tuned host–pathogen relationships are expected to mark their signature on the molecular make-up of the genes involved. However, molecular evolution of genes encoding immune response molecules of arthropod diseases vectors has received little attention, despite providing unique and valuable insights into the susceptibility to pathogens in other host species (Schlenke and Begun, 2003). Here, we describe and analyze polymorphism in the defensin gene within and between populations of the An. gambiae complex to address the following questions. Can selection be detected on this gene? If so, what mode of selection? And finally, do human pathogens mediate selection on defensin?

Defensin, a member of the cysteine-rich immune peptides, is a primary effector molecule produced by mosquitoes in response to infection with various pathogens (Richman et al., 1996). Defensin is synthesized mainly in the fat body of both larvae and adults and secreted into the haemolymph. It is expressed constitutively at low rates in adults and larvae, but following an infection challenge expression increases dramatically (Richman et al., 1997, Dimopoulos et al., 1998, Eggleston et al., 2000). Sporozoites of Plasmodium gallinaceum (and oocysts to a lower extent) are killed by defensin, but the relevance of this in vitro study to natural defense needs to be determined (Shahabuddin et al., 1998). Silencing of defensin in An. gambiae demonstrated that it is required for antimicrobial defense against Gram-positive bacteria (Blandin et al., 2002). Defensin is encoded by a single copy gene located at division 41 on the third chromosome of An. gambiae (Vizioli et al., 2001). It is comprised of two exons separated by a short intron. The 102 amino acids (aa) pre-pro-defensin includes a 25 aa signal peptide and a 77 aa segment that is cleaved to produce a 40 aa mature defensin. The signal and pro-peptide sequences of An. gambiae share little similarity with those from other insects, but the mature peptide is conserved and all insect defensins contain six cysteine residues. The promoter region is rich with sequence motifs similar to transcription regulatory elements of insect and mammalian immune response genes. These include binding sites for nuclear factor kappa B, GATA factors, nuclear factor interleukin 6 and interferon consensus elements among others (Eggleston et al., 2000). Induction of defensin transcription can be mediated by Gambif1 (Barillas-Mury et al., 1996), a member of the Rel protein group that mediates transcription regulation of immune response of Drosophila and other insects.

We chose populations from the highly anthropophilic species, An. gambiae, the moderately anthropophilic species, An. arabiensis, and the highly zoophilic species, An. quadriannulatus, representing high, moderate and no exposure to human pathogens, respectively (Hadis et al., 1997, Lemasson et al., 1997, Mouchet et al., 2004). The populations of An. gambiae and An. arabiensis are major vectors of Plasmodium falciparum with typical salivary glands infection rates of 3–9% (Hay et al., 2000, Mouchet et al., 2004 and references therein), but only the An. gambiae populations from Nigeria and eastern Kenya also transmit Wucheraria bancrofti, the causative agent of lymphatic filariasis (LF). The four An. gambiae populations include members of the M (Senegal) and S (West and East Kenya and Nigeria) molecular forms and span the maximal genetic distance measured among An. gambiae populations across the continent (Lehmann et al., 2003). Our study thus may help identify the potential and the limitation of such an approach for understanding the evolutionary forces that determine susceptibility to pathogens.