Gregory C. Lanzaro

Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics.

Closing the Vector Circle The genome sequence of Culex quinquefasciatus offers a representative of the third major genus of mosquito disease vectors for comparative analysis. In a major international effort, Arensburger et al. (p. 86) uncovered divergences in the C. quinquefasciatus genome compared with the representatives of the other two genera Aedes aegypti and Anopheles gambiae. The main difference noted is the expansion of numbers of genes, particularly for immunity, oxidoreductive functions, and digestive enzymes, which may reflect specific aspects of the Culex life cycle. Bartholomay et al. (p. 88) explored infection-response genes in Culex in more depth and uncovered 500 immune response-related genes, similar to the numbers seen in Aedes, but fewer than seen in Anopheles or the fruit fly Drosophila melanogaster. The higher numbers of genes were attributed partly to expansions in those encoding serpins, C-type lectins, and fibrinogen-related proteins, consistent with greater immune surveillance and associated signaling needed to monitor the dangers of breeding in polluted, urbanized environments. Transcriptome analysis confirmed that inoculation with unfamiliar bacteria prompted strong immune responses in Culex. The worm and virus pathogens that the mosquitoes transmit naturally provoked little immune activation, however, suggesting that tolerance has evolved to any damage caused by replication of the pathogens in the insects. The genome of a third mosquito species reveals distinctions related to vector capacities and habitat preferences. Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus: Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification.

Sandfly Maxadilan Exacerbates Infection with Leishmania major and Vaccinating Against It Protects Against L. major Infection

Bloodfeeding arthropods transmit many of the world’s most serious infectious diseases. Leishmania are transmitted to their mammalian hosts when an infected sandfly probes in the skin for a bloodmeal and injects the parasite mixed with its saliva. Arthropod saliva contains molecules that affect blood flow and modulate the immune response of the host. Indeed, sandfly saliva markedly enhances the infectivity of L. major for its host. If the salivary molecule(s) responsible for this phenomenon was identified, it might be possible to vaccinate the host against this molecule and thereby protect the host against infection with Leishmania. Such an approach represents a novel means of controlling arthropod-borne disease transmission. Here, we report that a single molecule, maxadilan, in sandfly saliva can exacerbate infection with L. major to the same degree as whole saliva, and that vaccinating against maxadilan protects mice against infection with L. major.

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae.

Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M‐ and S‐forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S‐form males and in 55 S‐form females we found one female containing M‐form sperm (1.82%). We also identified a mated M ×S hybrid adult female. From mating frequencies, we estimate the Nem between the M‐ and S‐form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow.

Sand Fly Saliva Enhances Leishmania amazonensis Infection by Modulating Interleukin-10 Production

ABSTRACT After transmission through the bite of female sand flies, Leishmania spp. can cause a broad spectrum of disease manifestations collectively known as leishmaniases. L. amazonensis is endemic in South America, where it causes cutaneous, diffuse cutaneous, and visceral leishmaniasis. In this study, we have provided evidence that salivary gland extracts (SGE) of Lutzomyia longipalpis enhances L. amazonensis infection. BALB/c mice infected intradermally in the ear with 105 metacyclic promastigotes of L. amazonensis together with SGE (equivalent to 0.5 gland) showed an early onset of disease and larger lesions that contained ∼3-log-units more parasites than did controls. To determine the potential mechanism underlying this enhancement, we assessed cytokine production via reverse transcriptase PCR and enzyme-linked immunosorbent assay. Mice coinjected with parasites and SGE displayed higher levels of interleukin-10 (IL-10) mRNA in the ear tissues, as well as higher levels of IL-10 in supernatants of restimulated draining lymph node (LN) cells, than did controls. Flow cytometric analysis revealed high frequencies of IL-10-producing CD4+ and CD8+ T cells in the draining LN of mice coinjected with the parasite and SGE. In addition, we examined bone marrow derived-macrophage cultures and detected increased IL-10 but decreased nitric oxide (NO) production in cells exposed to SGE prior to infection with L. amazonensis. Together, these results imply that the sand fly saliva facilitates Leishmania evasion of the host immune system by modulating IL-10 production.

Spatial and habitat distribution of Anopheles gambiae and Anopheles arabiensis (Diptera: Culicidae) in Banambani village, Mali.

Abstract We studied the larval distribution and composition of Anopheles arabiensis Patton, An. gambiae s.s. Giles, and its forms, among local habitats; and their association with the adults between these habitats in Banambani village, Mali during the mid-rainy seasons of 1997–1999. For species and form identification we used polymerase chain reaction (PCR) and PCR-restriction fragment-length polymorphism (RFLP). Differences among species in the distribution of larvae were observed in 1998, but not in 1997 or 1999, although they were on the borderline of statistical significance. Differences among the M and S molecular forms were statistically significant in 1999 when rainfall was high, but not in the two prior, drier sampling periods. Combining all information into the Fisher multiple comparisons test, there were statistically significant differences between species and molecular forms during the 3-yr study period. Hybrid larvae between the M and S forms were observed (0.57%), the first such observation to our knowledge. In spite of differences among larval distribution, no differences of adult species composition were observed among habitats. Factors that influence the distributions of An. gambiae larval populations are discussed.

Microsatellite DNA and isozyme variability in a West African population of Anopheles gambiae

Microsatellites are defined as tracts of tandemly repeated short DNA sequences. Polymorphisms in this class of DNA are currently being used to generate a genetic map of the mosquito Anopheles gambiae. In the present study we explore the potential of microsatellites as a tool for studying the genetic structure of natural populations of this malaria vector. Genetic polymorphism at twenty enzyme coding gene loci and eleven microsatellite DNA loci was surveyed in a population of An. gambiae from Mali, West Africa. All of the microsatellite loci surveyed were polymorphic, as compared to 40% of the isozyme loci. The mean heterozygosity for the isozyme loci was only 0.097 (±0.0035), but for the microsatellite loci it was 0.732 (±0.060). The pattern of variability was very different between isozymes and microsatellites. Typically, at an isozyme locus a single allele occurred at a frequency ≥0.75, whereas at microsatellite loci the most common allele had a frequency <0.50. We conclude that micro‐satellites provide a rich source of genetic polymorphisms for the study of the population genetics of An. gambiae and are in many ways superior to isozymes for this purpose. We discuss the potential for utilizing genetically mapped microsatellite loci to explore the effect of chromosomal inversions on the distribution of genetic polymorphisms in An. gambiae.

Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean-Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.

Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets

Significance We report that during a recent period of hybridization between two major African malaria mosquitoes, Anopheles gambiae and Anopheles coluzzii, an island of divergence on chromosome 2 introgressed from the A. gambiae into the A. coluzzii genome and its frequency subsequently increased. This introgression was coincident with the start of a major insecticide-treated bed net campaigns in Mali. These observations suggest that increased insecticide exposure acted as a selective force sufficient to drive introgression of an entire genomic island of divergence across the reproductive barrier separating these two species. This study provides a rare example of adaptive introgression in an animal species and elucidates the dynamics of how insecticide resistance evolved in A. coluzzii. Animal species adapt to changes in their environment, including man-made changes such as the introduction of insecticides, through selection for advantageous genes already present in populations or newly arisen through mutation. A possible alternative mechanism is the acquisition of adaptive genes from related species via a process known as adaptive introgression. Differing levels of insecticide resistance between two African malaria vectors, Anopheles coluzzii and Anopheles gambiae, have been attributed to assortative mating between the two species. In a previous study, we reported two bouts of hybridization observed in the town of Selinkenyi, Mali in 2002 and 2006. These hybridization events did not appear to be directly associated with insecticide-resistance genes. We demonstrate that during a brief breakdown in assortative mating in 2006, A. coluzzii inherited the entire A. gambiae-associated 2L divergence island, which includes a suite of insecticide-resistance alleles. In this case, introgression was coincident with the start of a major insecticide-treated bed net distribution campaign in Mali. This suggests that insecticide exposure altered the fitness landscape, favoring the survival of A. coluzzii/A. gambiae hybrids, and provided selection pressure that swept the 2L divergence island through A. coluzzii populations in Mali. We propose that the work described herein presents a unique description of the temporal dynamics of adaptive introgression in an animal species and represents a mechanism for the rapid evolution of insecticide resistance in this important vector of human malaria in Africa.

Microsatellite DNA Polymorphism and Heterozygosity Among Field and Laboratory Populations of Anopheles gambiae s.s. (Diptera: Culicidae)

Abstract We compared microsatellite polymorphism at nine loci located on chromosome 3 among two colonies and a field population of Anopheles gambiae sensu stricto Giles mosquitoes. Numbers of microsatellite alleles observed at each locus and mean heterozygosities were drastically reduced among laboratory colonies. Genetic analysis of the field population used in this study revealed an unprecedented frequency of rare alleles (<0.05). In contrast, colony samples revealed large numbers of alleles with frequencies >0.50. Partitioning of field data to assess the impact of rare alleles, null alleles, and sample size on estimates of mean heterozygosity revealed the plasticity of this measurement and suggests that heterozygosity may be reliably estimated from relatively small collections using microsatellites.

Relationship Between kdr Mutation and Resistance to Pyrethroid and DDT Insecticides in Natural Populations of Anopheles gambiae

Abstract The spread of insecticide resistance genes in Anopheles gambiae Giles sensu stricto threatens to compromise vector-based malaria control programs. Two mutations at the same locus in the voltage-gated sodium channel gene are known to confer knockdown resistance (kdr) to pyrethroids and DDT. Kdr-e involves a leucine-serine substitution, and it was until recently thought to be restricted to East Africa, whereas kdr-w, which involves a leucine-phenylalanine substitution, is associated with resistance in West Africa. In this study, we analyze the frequency and relationship between the kdr genotypes and resistance to type I and type II pyrethroids and DDT by using WHO test kits in both the Forest-M and S molecular forms of An. gambiae in Cameroon. Both kdr-w and kdr-e polymorphisms were found in sympatric An. gambiae, and in many cases in the same mosquito. Kdr-e and kdr-w were detected in both forms, but they were predominant in the S form. Both kdr-e and kdr-w were closely associated with resistance to DDT and weakly associated with resistance to type II pyrethroids. Kdr-w conferred greater resistance to permethrin than kdr-e. We also describe a modified diagnostic designed to detect both resistant alleles simultaneously.