Mapping quantitative trait loci in noninbred mosquito crosses.

The identification of genes that affect quantitative traits has been of great interest to geneticists for many decades, and many statistical methods have been developed to map quantitative trait loci (QTL). Most QTL mapping studies in experimental organisms use purely inbred lines, where the two homologous chromosomes in each individual are identical. As a result, many existing QTL mapping methods developed for experimental organisms are applicable only to genetic crosses between inbred lines. However, it may be difficult to obtain inbred lines for certain organisms, e.g., mosquitoes. Although statistical methods for QTL mapping in outbred populations, e.g., humans, can be applied for such crosses, these methods may not fully take advantage of the uniqueness of these crosses. For example, we can generally assume that the two grandparental lines are homozygous at the QTL of interest, but such information is not be utilized through methods developed for outbred populations. In addition, mating types and phases can be relatively easy to establish through the analysis of adjacent markers due to the large number of offspring that can be collected, substantially simplifying the computational need. In this article, motivated by a mosquito intercross experiment involving two selected lines that are not genetically homozygous across the genome, we develop statistical methods for QTL mapping for genetic crosses involving noninbred lines. In our procedure, we first infer parental mating types and use likelihood-based methods to infer phases in each parent on the basis of genotypes of offspring and one parent. A hidden Markov model is then employed to estimate the number of high-risk alleles at marker positions and putative QTL positions between markers in each offspring, and QTL mapping is finally conducted through the inferred QTL configuration across all offspring in all crosses. The performance of the proposed methods is assessed through simulation studies, and the usefulness of this method is demonstrated through its application to a mosquito data set.

Biology of Toll receptors: lessons from insects and mammals.

Toll receptors are type I transmembrane proteins that play important roles in development and immunity in animals. Comparison of the genomes of mouse and human on one side and of the fruitfly Drosophila and the mosquito Anopheles (two dipteran insects) on the other, revealed that the four species possess a similar number of Toll receptors (approximately 10). However, phylogenetic analyses indicate that the families of Toll receptors expanded independently in insects and mammals. We review recent results on these receptors, which point to differences in the activation and signaling between Tolls in insects and Toll-like receptors (TLRs) in mammals. Whereas mammalian TLRs appear to be solely dedicated to host-defense, insect Tolls may be predominantly linked to other functions, probably developmental.

The mosquito genome--a turning point?

Of the insects that serve as vectors for parasitic diseases, the genus Anopheles is the most important. Of the approximately 400 species, about twelve serve as vectors for human pathogens. Months have passed since the sequenced genomes of the malaria parasite, Plasmodium falciparum, and its vector, Anopheles gambiae, were published. Sequences were compared, gene and protein predictions were made, new research areas evolved and many ongoing projects gained new momentum. A general belief is that we are at a turning point: we are now in a position to tackle both the parasite and the vector from new angles and with new force, for example, by identifying new drug targets and obtaining a deeper insight into molecular mechanisms of the insect, the parasite and the interactions between them.

Characterization of four Toll related genes during development and immune responses in Anopheles gambiae.

Toll-like receptors (TLRs) are a group of evolutionary conserved proteins with diverse biological functions. In Drosophila melanogaster, Toll protein plays an important role in pattern formation in embryogenesis and in antimicrobial immunity in larvae and adults. In insects, Toll and two other related proteins, Tehao and 18-wheeler have been shown to participate in the activation of the innate immune responses to fungal and bacterial pathogens. In this paper we report the cloning and characterization of four TLR gene from malaria vector mosquito Anopheles gambiae, AgToll, AgToll6, AgTrex, and AgToll9, orthologues of DmToll, DmToll6, DmTollo (Toll8) and DmToll9 (CG5528) in Drosophila melanogaster. The expression profiles of these genes during development, in different adult tissues and after immune challenge were examined. As expected for the orthologue of Drosophila Toll, AgToll was found to be expressed highly in the ovary and may play a role in pattern formation during embryogenesis. AgToll9, surprisingly, was found to be highly expressed in the adult gut. The potential roles of these genes in development and immunity were discussed.

Quantitative trait loci in Anopheles gambiae controlling the encapsulation response against Plasmodium cynomolgi Ceylon.

BACKGROUND: Anopheles gambiae females are the world's most successful vectors of human malaria. However, a fraction of these mosquitoes is refractory to Plasmodium development. L3-5, a laboratory selected refractory strain, encapsulates transforming ookinetes/early oocysts of a wide variety of Plasmodium species. Previous studies on these mosquitoes showed that one major (Pen1) and two minor (Pen2, Pen3) autosomal dominant quantitative trait loci (QTLs) control the melanotic encapsulation response against P. cynomolgi B, a simian malaria originating in Malaysia. RESULTS: We have investigated the response of L3-5 to infection with P. cynomolgi Ceylon, a different but related parasite species, in crosses with the susceptible strain 4Arr. Refractoriness to this parasite is incompletely recessive. Infection and genotyping of F2 intercross females at genome-spanning microsatellite loci revealed that 3 autosomal QTLs control encapsulation of this species. Two loci map to the regions containing Pen2 and Pen3. The novel QTL maps to chromosome 3R, probably to polytene division 32 or 33. Thus the relative contribution of any QTL to oocyst encapsulation varies with the species of parasite. Further, different QTLs were most readily identified in different F2 families. This, like the F1 data, suggests that L3-5 is not genetically homogeneous and that somewhat different pathways may be used to achieve an encapsulation response. CONCLUSION: We have shown here that different QTLs are involved in responses against different Plasmodium parasites.

Mosaic genome architecture of the Anopheles gambiae species complex.

BACKGROUND: Attempts over the last three decades to reconstruct the phylogenetic history of the Anopheles gambiae species complex have been important for developing better strategies to control malaria transmission. METHODOLOGY: We used fingerprint genotyping data from 414 field-collected female mosquitoes at 42 microsatellite loci to infer the evolutionary relationships of four species in the A. gambiae complex, the two major malaria vectors A. gambiae sensu stricto (A. gambiae s.s.) and A. arabiensis, as well as two minor vectors, A. merus and A. melas. PRINCIPAL FINDINGS: We identify six taxonomic units, including a clear separation of West and East Africa A. gambiae s.s. S molecular forms. We show that the phylogenetic relationships vary widely between different genomic regions, thus demonstrating the mosaic nature of the genome of these species. The two major malaria vectors are closely related and closer to A. merus than to A. melas at the genome-wide level, which is also true if only autosomes are considered. However, within the Xag inversion region of the X chromosome, the M and two S molecular forms are most similar to A. merus. Near the X centromere, outside the Xag region, the two S forms are highly dissimilar to the other taxa. Furthermore, our data suggest that the centromeric region of chromosome 3 is a strong discriminator between the major and minor malaria vectors. CONCLUSIONS: Although further studies are needed to elucidate the basis of the phylogenetic variation among the different regions of the genome, the preponderance of sympatric admixtures among taxa strongly favor introgression of different genomic regions between species, rather than lineage sorting of ancestral polymorphism, as a possible mechanism.

Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes.

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae.

We show that, in the malaria vector Anopheles gambiae, expression of Cecropin 1 is regulated by REL2, an NF-kappaB-like transcription factor orthologous to Drosophila Relish. Through alternative splicing, REL2 produces a full-length (REL2-F) and a shorter (REL2-S) protein isoform lacking the inhibitory ankyrin repeats and death domain. RNA interference experiments show that, in contrast to Drosophila Relish, which responds solely to Gram-negative bacteria, the Anopheles REL2-F and REL2-S isoforms are involved in defense against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria, respectively. REL2-F also regulates the intensity of mosquito infection with the malaria parasite, Plasmodium berghei. The adaptor IMD shares the same activities as REL2-F. Microarray analysis identified 10 additional genes regulated by REL2, including CEC3, GAM1, and LRIM1.

Origin of Toll-like receptor-mediated innate immunity.

Toll-related receptors (TLR) have been found in four animal phyla: Nematoda, Arthropoda, Echinodermata, and Chordata. No TLR has been identified thus far in acoelomates. TLR genes play a pivotal role in the innate immunity in both fruit fly and mammals. The prevailing view is that TLR-mediated immunity is ancient. The two pseudocoelomate TLRs, one each from Caenorhabditis elegans and Strongyloides stercoralis, were distinct from the coelomate ones. Further, the only TLR gene (Tol-1) in Ca. elegans did not appear to play a role in innate immunity. We argue that TLR-mediated innate immunity developed only in the coelomates, after they split from pseudocoelomates and acoelomates. We hypothesize that the function of TLR-mediated immunity is to prevent microbial infection in the body cavity present only in the coelomates. Phylogenetic analysis showed that almost all arthropod TLRs form a separate cluster from the mammalian counterparts. We further hypothesize that TLR-mediated immunity developed independently in the protostomia and deuterostomia coelomates.

Immunity-related genes and gene families in Anopheles gambiae.

We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.