Xin-zhuan Su and colleagues report genome-wide SNP genotyping of 189 culture-adapted Plasmodium falciparum parasites and examine population structure, selection and recombination hotspots. They also conduct genome-wide association studies for resistance to seven different antimalarial drugs. Antimalarial drugs impose strong selective pressure on Plasmodium falciparum parasites and leave signatures of selection in the parasite genome1,2; screening for genes under selection may suggest potential drug or immune targets3. Genome-wide association studies (GWAS) of parasite traits have been hampered by the lack of high-throughput genotyping methods, inadequate knowledge of parasite population history and time-consuming adaptations of parasites to in vitro culture. Here we report the first Plasmodium GWAS, which included 189 culture-adapted P. falciparum parasites genotyped using a custom-built Affymetrix molecular inversion probe 3K malaria panel array with a coverage of ∼1 SNP per 7 kb. Population structure, variation in recombination rate and loci under recent positive selection were detected. Parasite half-maximum inhibitory concentrations for seven antimalarial drugs were obtained and used in GWAS to identify genes associated with drug responses. This study provides valuable tools and insight into the P. falciparum genome.

The human malaria parasite Plasmodium falciparum survives pressures from the host immune system and antimalarial drugs by modifying its genome. Genetic recombination and nucleotide substitution are the two major mechanisms that the parasite employs to generate genome diversity. A better understanding of these mechanisms may provide important information for studying parasite evolution, immune evasion and drug resistance. Here, we used a high-density tiling array to estimate the genetic recombination rate among 32 progeny of a P. falciparum genetic cross (7G8 × GB4). We detected 638 recombination events and constructed a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb per centimorgan and identified 54 candidate recombination hotspots. Similar to centromeres in other organisms, the sequences of P. falciparum centromeres are found in chromosome regions largely devoid of recombination activity. Motifs enriched in hotspots were also identified, including a 12-bp G/C-rich motif with 3-bp periodicity that may interact with a protein containing 11 predicted zinc finger arrays. These results show that the P. falciparum genome has a high recombination rate, although it also follows the overall rule of meiosis in eukaryotes with an average of approximately one crossover per chromosome per meiosis. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination rate observed. The lack of recombination activity in centromeric regions is consistent with the observations of reduced recombination near the centromeres of other organisms.

Background: In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. Results: Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein. Conclusion: This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the sand fly Lutzomyia longipalpis. This analysis provides the detailed information of molecules present in the midgut of this sand fly and the transcripts potentially modulated by blood feeding and by the presence of the Leishmania parasite. More importantly, this analysis suggests that Leishmania infantum chagasi alters the expression profile of certain midgut transcripts in the sand fly during blood meal digestion and that this modulation may be relevant for the survival and establishment of the parasite in the gut of the fly. Moreover, this analysis suggests that these changes may be occurring during the digestion of the blood meal and not afterwards.

Immunization with the highly polymorphic Plasmodium falciparum apical membrane antigen 1 ( Pf AMA1) induces protection in animals but primarily against parasites that express the same or similar alleles. One strategy to overcome the obstacle of polymorphism is to combine Pf AMA1 proteins representing major haplotypes into one vaccine. To determine the minimum number of haplotypes that would confer broad protection, we sequenced the coding region of PfAMA1 from 97 clones from around the world and 61 isolates from Mali, identifying 150 haplotypes for domains 1 to 3 that included previous sequences. A clustering algorithm grouped the 150 haplotypes into six populations that were independent of geographic location. Each of the six populations contained haplotypes predominantly of that population (predominant haplotypes) and haplotypes that were a mixture of haplotypes represented in other populations (admixed haplotypes). To determine the biological relevance of the populations identified through the clustering algorithm, antibodies induced against one predominant haplotype of population 1 (3D7) and one admixed haplotype of population 5 (FVO) were tested for their ability to block parasite invasion of erythrocytes. Parasites expressing Pf AMA1s belonging to population 1 were efficiently inhibited by 3D7-specific antibodies, whereas parasites expressing Pf AMA1s belonging to other populations were not. For FVO-specific antibodies, we observed growth inhibition against itself as well as isolates belonging to populations 3 and 6. Our data suggests that the inclusion of Pf AMA1 sequences from each of the six populations may result in a vaccine that induces protective immunity against a broad range of malaria parasites.

Background: Sand fly saliva plays an important role in blood feeding andLeishmaniatransmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused onPhlebotomus (Adlerius) arabicus, which was recently shown to transmitLeishmania tropica, the causative agent of cutaneous leishmaniasis in Israel.Results: A cDNA library from salivary glands ofP. arabicusfemales was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten byP. arabicusto salivary antigens was assessed and many salivary proteins were determined to be antigenic.Conclusion: This transcriptomic analysis ofP. arabicussalivary glands is the first description of salivary proteins of a sand fly in the subgenusAdlerius. Proteomic analysis ofP. arabicussalivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-Leishmaniainteractions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.

In recent years, there have been several sialome projects revealing transcripts expressed in the salivary glands of ticks, which are important vectors of several human diseases. Here, we focused on the sialome of the European vector of Lyme disease, Ixodes ricinus.