Douglas E. Norris

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.

Mosquito-borne Diseases as a Consequence of Land Use Change

Human modification of the natural environment continues to create habitats in which mosquitoes, vectors of a wide variety of human and animal pathogens, thrive if unabated with an enormous potential to negatively affect public health. Historic examples of these modifications include of impoundments, dams, and irrigation systems that create havens for the mosquitoes that transmit malaria, dengue, and filariasis. Additionally, contemporary deforestation appears to be associated with the expansion of mosquito distributions and the increase in mosquito-borne disease transmission. These observations are not unique to the developing world, as urban sprawl also contributes significantly to mosquito habitats and offers a sanctuary to some vector populations. With foresight and planning, most of these systems can be appropriately managed to control vector populations and pathogen transmission. The key to disease control is developing an understanding of the contribution of human landscape modification to vector-borne pathogen transmission and how a balance may be achieved between human development, public health, and responsible land use.

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.

Analysis of Anopheles arabiensis Blood Feeding Behavior in Southern Zambia during the Two Years after Introduction of Insecticide-Treated Bed Nets

Anopheles arabiensis mosquitoes are the primary vector responsible for Plasmodium falciparum transmission in Macha, Zambia. Because insecticide-treated bed nets (ITNs) have the potential to alter host feeding behavior, the extent of the zoophilic and exophagic tendencies of the vector was evaluated during the two rainy seasons after ITN introduction. Centers for Disease Control light traps, paired indoor/outdoor human landing catches, and outdoor cattle-baited collections were used to assess potential changes in host preference. Results support the hypothesis that An. arabiensis mosquitoes in Macha remain highly anthropophilic despite high ITN use. Anopheles arabiensis mosquitoes in Macha appear to be relatively exophagic and have been caught biting outdoors immediately after sunset and before sunrise, potentially circumventing some of the protective effects of ITNs.

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.

Commensal ecology, urban landscapes, and their influence on the genetic characteristics of city-dwelling Norway rats (Rattus norvegicus).

Movement of individuals promotes colonization of new areas, gene flow among local populations, and has implications for the spread of infectious agents and the control of pest species. Wild Norway rats (Rattus norvegicus) are common in highly urbanized areas but surprisingly little is known of their population structure. We sampled individuals from 11 locations within Baltimore, Maryland, to characterize the genetic structure and extent of gene flow between areas within the city. Clustering methods and a neighbour‐joining tree based on pairwise genetic distances supported an east–west division in the inner city, and a third cluster comprised of historically more recent sites. Most individuals (~95%) were assigned to their area of capture, indicating strong site fidelity. Moreover, the axial dispersal distance of rats (62 m) fell within typical alley length. Several rats were assigned to areas 2–11.5 km away, indicating some, albeit infrequent, long‐distance movement within the city. Although individual movement appears to be limited (30–150 m), locations up to 1.7 km are comprised of relatives. Moderate FST, differentiation between identified clusters, and high allelic diversity indicate that regular gene flow, either via recruitment or migration, has prevented isolation. Therefore, ecology of commensal rodents in urban areas and life‐history characteristics of Norway rats likely counteract many expected effects of isolation or founder events. An understanding of levels of connectivity of rat populations inhabiting urban areas provides information about the spatial scale at which populations of rats may spread disease, invade new areas, or be eradicated from an existing area without reinvasion.

Borrelia burgdorferi ospC Heterogeneity among Human and Murine Isolates from a Defined Region of Northern Maryland and Southern Pennsylvania: Lack of Correlation with Invasive and Noninvasive Genotypes

ABSTRACT B. burgdorferi invasiveness correlates with ospC genotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites, B. burgdorferi ospC heterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patients home were isolated. 3′ ospC sequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same ospC were seen in only one locality, and five of six sites contained two or more B. burgdorferi ospC clones. Four invasive patient isolates and six small mammal isolates clustered with “noninvasive” reference ospC genotypes. A high degree of ospC diversity exists among B. burgdorferi isolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive ospC types contradicts the ospC invasiveness hypothesis. Alternative genetic markers for B. burgdorferi disseminated disease should be investigated.

In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy

ABSTRACT Clinical treatment failures have been reported to occur in early Lyme borreliosis (LB) for many suitable antimicrobial agents. Investigations of possible resistance mechanisms of the Borrelia burgdorferi complex must analyze clinical isolates obtained from LB patients, despite their receiving antibiotic treatment. Here, borrelial isolates obtained from five patients with erythema migrans (EM) before the start of antibiotic therapy and again after the conclusion of treatment were investigated. The 10 isolates were characterized by restriction fragment length polymorphism analysis and plasmid profile analysis and subjected to susceptibility testing against a variety of antimicrobial agents including those used for initial chemotherapy. Four out of five patients were infected by the same genospecies (Borrelia afzelii, n = 3; Borrelia garinii, n = 1) at the site of the EM lesion before and after antimicrobial therapy. In one patient the genospecies of the initial isolate (B. afzelii) differed from that of the follow-up isolate (B. garinii). No significant changes in the in vitro susceptibilities became obvious for corresponding clinical isolates before the start and after the conclusion of antimicrobial therapy. This holds true for the antimicrobial agents used for specific chemotherapy of the patients, as well as for any of the additional agents tested in vitro. Our study substantiates borrelial persistence in some EM patients at the site of the infectious lesion despite antibiotic treatment over a reasonable time period. Borrelial persistence, however, was not caused by increasing MICs or minimal borreliacidal concentrations in these isolates. Therefore, resistance mechanisms other than acquired resistance to antimicrobial agents should be considered in patients with LB resistant to treatment.

The taxonomic status of genetically divergent populations of Lutzomyia longipalpis (Diptera: Psychodidae) based on the distribution of mitochondrial and isozyme variation.

Abstract The sand fly, Lutzomyia longipalpis (Lutz & Neiva) reputedly is a complex of cryptic species; however, there is currently no consensus as to the number of species in the complex or their geographic distributions. We conducted phylogenetic analyses of 31 populations from throughout the species range, using seven isozyme loci and genes in the mitochondrial genome. Analyses of these two independent sets of markers were largely concordant and revealed four distinct clades that support the existence of four species. The four clades have distinct geographic ranges: (1) Brazil (Species A = Lu. longipalpis sensu stricto), (2) Laran (Species B = Lu. pseudolongipalpis), (3) cis-Andean (Species C), and (4) trans-Andean (Species D). The cis-Andean clade may be subdivided further into two groups, one in Colombia and one in northwestern Venezuela, but their taxonomic status remains unresolved. Knowledge that Lu. longipalpis is a complex of species may ultimately shed light on anomalies in the epidemiology of visceral leishmaniasis in the New World.

Spotted-Fever Group Rickettsia in Dermacentor variabilis, Maryland

Three-hundred ninety-two adult Dermacentor variabilis were collected from six Maryland counties during the spring, summer, and fall of 2002. Infection prevalence for spotted fever group Rickettsia was 3.8%, as determined by polymerase chain reaction. Single strand conformational polymorphism (SSCP) analysis followed by sequencing indicated that all infections represented a single rickettsial taxon, Rickettsia montanensis.