Patterns of Selection in Anti-Malarial Immune Genes in Malaria Vectors: Evidence for Adaptive Evolution in LRIM1 in Anopheles arabiensis Michel A. Slotman 1 *, Aristeidis Parmakelis 1 , Jonathon C. Marshall 1¤ , Parfait H. Awono-Ambene 2 , Christophe Antonio-Nkondjo 2 , Frederic Simard 2,3 , Adalgisa Caccone 1 , Jeffrey R. Powell 1 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, United States of America, 2 Organisation de Coordination pour la Lutte Contre les Ende ´ mies en Afrique Centrale, Yaounde ´ , Cameroon, 3 Institute de Recherche pour le De ´ veloppement, Yaounde ´, Cameroon Background. Co-evolution between Plasmodium species and its vectors may result in adaptive changes in genes that are crucial components of the vector’s defense against the pathogen. By analyzing which genes show evidence of positive selection in malaria vectors, but not in closely related non-vectors, we can identify genes that are crucial for the mosquito’s resistance against Plasmodium. Methodology/Principle Findings. We investigated genetic variation of three anti-malarial genes; CEC1, GNBP-B1 and LRIM1, in both vector and non-vector species of the Anopheles gambiae complex. Whereas little protein differentiation was observed between species in CEC1 and GNBP-B1, McDonald-Kreitman and maximum likelihood tests of positive selection show that LRIM1 underwent adaptive evolution in a primary malaria vector; An. arabiensis. In particular, two adjacent codons show clear signs of adaptation by having accumulated three out of four replacement substitutions. Furthermore, our data indicate that this LRIM1 allele has introgressed from An. arabiensis into the other main malaria vector An. gambiae. Conclusions/Significance. Although no evidence exists to link the adaptation of LRIM1 to P. falciparum infection, an adaptive response of a known anti-malarial gene in a primary malaria vector is intriguing, and may suggest that this gene could play a role in Plasmodium resistance in An. arabiensis. If so, our data also predicts that LRIM1 alleles in An. gambiae vary in their level of resistance against P. falciparum. Citation: Slotman MA, Parmakelis A, Marshall JC, Awono-Ambene PH, Antonio-Nkondjo C, et al (2007) Patterns of Selection in Anti-Malarial Immune Genes in Malaria Vectors: Evidence for Adaptive Evolution in LRIM1 in Anopheles arabiensis. PLoS ONE 2(8): e793. doi:10.1371/journal.pone.0000793 INTRODUCTION Despite ongoing control efforts during the last decades, malaria remains one of the most deadly infectious diseases. The vast majority of its burden is carried by people on the African continent, where 1 to 2 million people die annually from this disease [1]. Current malaria control efforts are hampered by the spread of insecticide and drug resistance, which has inspired research programs aimed at the development and eventual release of genetically altered mosquitoes that would be resistant to Plasmodium falciparum transmission. The need to identify refractory genes for this effort has focused much attention on the immune system of malaria’s main vector in Africa, An. gambiae. The completion of the An. gambiae genome [2] has greatly facilitated research in this direction, and various anti-malarial immunity genes have now been identified [e.g. 3–6]. Additionally, two recent studies provided many candidate anti-malarial immune genes that are up-regulated in response to Plasmodium infection [7,8]. So far little attention has been devoted to examining poly- morphism of immunity genes in natural malaria vector popula- tions [9,10]. It is known however that molecules that are involved in interactions with pathogens, such as immune genes, are one of the major types of proteins on which positive selection has been demonstrated [11,12]. Presumably, this is because such genes are involved in co-evolution between hosts and pathogens. In the case of malaria, if Plasmodium infection affects the mosquito’s fitness, we may expect the accumulation of adaptive amino acid substitutions in those anti-malarial genes that are crucial in specifically limiting Plasmodium infection in vector species, whereas such changes should not be found in closely related species that do not transmit malaria. That An. gambiae has in fact undergone an adaptive response to P. falciparum infection is suggested by several lines of evidence. First of all, P. falciparum goes through severe bottlenecks during its life cycle in this mosquito [13], demonstrating that the mosquito immune system is limiting the Plasmodium infection. Furthermore, P. berghei, which is not transmitted naturally by An. gambiae, produces a much higher oocyst number in An. gambiae than its natural pathogen P. falciparum. In fact, in a review of studies estimating the fitness effect of Plasmodium infection on Anopheles species, reduced fitness was observed in 10 combinations of Plasmodium and Anopheles species that do not occur naturally, whereas in 10 natural combinations, including An. gambiae and P. falciparum, no fitness effects were observed [14]. This is an indication that Anopheles species have evolved to limit infections of the Plasmodium species they come into contact with. This is corroborated by the fact that the immune response to P. falciparum Academic Editor: Leah Cowen, University of Toronto, Canada Received June 29, 2007; Accepted July 18, 2007; Published August 29, 2007 Copyright: ß 2007 Slotman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by National Institutes of Health grant RO1 A1 046018 to JRP. Additionally, AP was supported by a Marie Curie Outgoing International Fellowship (Contract No. MOIF-CT-2006-021357). Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: michel.slotman@yale. edu ¤ Current address: Department of Biology, Southern Utah University, Cedar City, Utah, United States of America PLoS ONE | www.plosone.org 1 August 2007 | Issue 8 | e793